Methods for producing Fabs and bi-specific antibodies

ABSTRACT

The present invention provides methods for producing Fabs and bi-specific antibodies comprising designed residues in the interfaces of the heavy chain-light chain variable (V H /V L ) domain and the heavy chain-light chain constant (C H1 /C L ) domain, Fabs and bi-specific antibodies produced according to said processes and host cells encoding the same.

Antibody therapies represent an ever increasing segment of the globalpharmaceutical market. Approved antibody-based products includetreatments for cancer, rheumatoid arthritis, infectious diseases,cardiovascular disease and autoimmune disorders. However, to improvepatient outcomes, co-administration of two or more agents that perturbdistinct therapeutic targets or biochemical pathways is often desired.In this context, antibody therapy has limitations.

Co-administration of two or more antibody therapies requires multipleinjections or, alternatively, a single injection of a co-formulation oftwo different antibody compositions. While multiple injections permitflexibility in dose and timing of administration, the inconvenience anddiscomfort associated with multiple injections may reduce patientcompliance. On the other hand, while a co-formulation of multipleantibody agents would permit fewer injections, the difficulty and/orexpense associated with designing a suitable pharmaceutical formulationthat provides the necessary stability and bioavailability, for eachantibody ingredient, may be prohibitive. Furthermore, any treatmentregime which entails administration of separate antibody agents willincur the added manufacturing and regulatory cost associated with thedevelopment of each individual agent.

The archetypical antibody is comprised of two identical antigen bindingfragments (Fabs) which not only direct binding to a particular antigenicdeterminant, but also provide the interface for assembly between heavychain (HC)-light chain (LC) pairs. Bispecific antibodies—single agentscapable of binding to two distinct antigens—have been proposed as ameans for addressing the limitations attendant with co-administration orco-formulation of separate antibody agents. Bispecific antibodies mayintegrate the binding activity of two separate MAb therapeutics,providing a cost and convenience benefit to the patient. Under certaincircumstances, bispecific antibodies may elicit synergistic or novelactivities beyond what an antibody-combination can achieve. One exampleof novel activity provided by bispecific antibodies would be thebridging of two different cell types through the binding of distinctcell surface receptors. Alternately, bispecific antibodies couldcross-link two receptors on the surface of the same cell leading tonovel agonistic/antagonistic mechanisms.

The ability to generate bispecific antibodies with fully IgGarchitecture has been a long-standing challenge in antibody engineering.One proposal for generating fully IgG bispecific antibodies entailsco-expression of nucleic acids encoding two, distinct HC-LC pairs which,when expressed, assemble to form a single antibody comprising twodistinct Fabs. However, challenges with this approach remain.Specifically, the expressed polypeptides of each desired Fab mustassemble with good specificity to reduce generation of mis-matchedbyproducts, and the resulting heterotetramer must assemble with goodstability. Procedures for directing assembly of particular HC-HC pairsby introducing modifications into regions of the HC-HC interface havebeen disclosed in the art. (See Klein et al., mAbs; 4(6); 1-11 (2012);Carter et al., J. Immunol. Methods; 248; 7-15 (2001); Gunasekaran, etal., J. Biol. Chem.; 285; 19637-19646 (2010); Zhu et al., Protein Sci.;6: 781-788 (1997); and Igawa et al., Protein Eng. Des. Sel.; 23; 667-677(2010)). However, there remains a need for alternative methods.

In accordance with the present invention, methods have been identifiedfor achieving assembly of particular Fabs by co-expressing nucleic acidsencoding particular HC-LC pairs which contain designed residues in theinterface of the heavy chain-light chain variable (V_(H)/V_(L)) domainsand the heavy chain-light chain constant (C_(H1)/C_(L)) domains. Moreparticularly, the methods of the present invention achieve improvedspecificity and, or stability in assembly of particular Fabs. Even moreparticular, the methods of the present invention allow the bindingspecificities and binding activities of the variable regions of twodistinct therapeutic antibodies to be combined in a single bi-specificantibody compound.

Thus, the present invention provides a method for producing a fragment,antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a)a first nucleic acid encoding both a heavy chain variable domain and anIgG heavy chain constant CH1 domain, wherein said heavy chain variabledomain comprises a lysine substituted at residue 39 (39K) and a glutamicacid substituted at the residue which is four amino acids upstream ofthe first residue of HFR3 according to Kabat, and said heavy chain CH1domain comprises an alanine substituted at residue 172 (172A) and aglycine substituted at residue 174 (174G); and (b) a second nucleic acidencoding both a light chain variable domain and a light chain constantdomain wherein said light chain variable domain is a kappa isotype andcomprises an arginine substituted at residue 1 (1R) and an aspartic acidsubstituted at residue 38 (38D), and said light chain constant domaincomprises a tyrosine substituted at residue 135 (135Y) and a tryptophansubstituted at residue 176 (176W), wherein each of said heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to the same antigen; (2) cultivatingsaid host cell under conditions such that said heavy chain variable andconstant domains and said light chain variable and constant domains areproduced; and (3) recovering from said host cell a Fab comprising saidheavy chain variable and constant domains and said light chain variableand constant domains. More particular to this embodiment, the presentinvention provides a method comprising one or more of the following:said first nucleic acid encodes a heavy chain CH1 constant domainfurther comprising a methionine or isoleucine substituted at residue 190(190M or 190I); said second nucleic acid encodes a light chain constantdomain further comprising a leucine substituted at residue 133 (133L);and said second nucleic acid encodes a light chain constant domainfurther comprising a glutamine or aspartic acid substituted at residue174 (174Q or 174D).

In a separate embodiment, the present invention provides a method forproducing a fragment, antigen binding (Fab) comprising: (1)co-expressing in a host cell: (a) a first nucleic acid encoding both aheavy chain variable domain and an IgG heavy chain constant CH1 domain,wherein said heavy chain variable domain comprises a lysine substitutedat residue 39 (39K) and a glutamic acid substituted at the residue whichis four amino acids upstream of the first residue of HFR3 according toKabat, and said heavy chain CH1 domain comprises an arginine substitutedat residue 172 (172R) and a glycine substituted at residue 174 (174G);and (b) a second nucleic acid encoding both a light chain variabledomain and a light chain constant domain wherein said light chainvariable domain is a kappa isotype and comprises an arginine substitutedat residue 1 (1R) and an aspartic acid substituted at residue 38 (38D),and said light chain constant domain comprises a tyrosine substituted atresidue 135 (135Y) and a tryptophan substituted at residue 176 (176W),wherein each of said heavy chain and light chain variable domainscomprise three complementarity determining regions (CDRs) which directbinding to the same antigen; (2) cultivating said host cell underconditions such that said heavy chain variable and constant domains andsaid light chain variable and constant domains are produced; and (3)recovering from said host cell a Fab comprising said heavy chainvariable and constant domains and said light chain variable and constantdomains. More particular to this embodiment, the present inventionprovides a method comprising one or more of the following: said firstnucleic acid encodes a heavy chain CH1 constant domain furthercomprising a methionine or isoleucine substituted at residue 190 (190Mor 190I); said second nucleic acid encodes a light chain constant domainfurther comprising a leucine substituted at residue 133 (133L); and saidsecond nucleic acid encodes a light chain constant domain furthercomprising a glutamine or aspartic acid substituted at residue 174 (174Qor 174D).

In another embodiment, the present invention provides a method forproducing a fragment, antigen binding (Fab) comprising: (1)co-expressing in a host cell: (a) a first nucleic acid encoding both aheavy chain variable domain and an IgG heavy chain constant CH1 domain,wherein said heavy chain variable domain comprises a lysine substitutedat residue 39 (39K) and a glutamic acid substituted at the residue whichis four amino acids upstream of the first residue of HFR3 according toKabat, and said heavy chain CH1 domain comprises an alanine substitutedat residue 172 (172A) and a glycine substituted at residue 174 (174G);and (b) a second nucleic acid encoding both a light chain variabledomain and a light chain constant domain wherein said light chainvariable domain is a kappa isotype and comprises an arginine substitutedat residue 1 (1R) and an aspartic acid substituted at residue 38 (38D),and said light chain constant domain comprises a phenylalaninesubstituted at residue 135 (135F) and a tryptophan substituted atresidue 176 (176W), wherein each of said heavy chain and light chainvariable domains comprise three complementarity determining regions(CDRs) which direct binding to the same antigen; (2) cultivating saidhost cell under conditions such that said heavy chain variable andconstant domains and said light chain variable and constant domains areproduced; and (3) recovering from said host cell a Fab comprising saidheavy chain variable and constant domains and said light chain variableand constant domains.

In another embodiment, the present invention provides a method forproducing a fragment, antigen binding (Fab) comprising; (1)co-expressing in a host cell: (a) a first nucleic acid encoding both aheavy chain variable domain and an IgG heavy chain constant CH1 domain,wherein said heavy chain variable domain comprises a tyrosinesubstituted at residue 39 (39Y) and said heavy chain CH1 domaincomprises a WT sequence; and (b) a second nucleic acid encoding both alight chain variable domain and a light chain constant domain whereinsaid light chain variable domain comprises an arginine substituted atresidue 38 (38R) and said light chain constant domain comprises a WTsequence, wherein each of said heavy chain and light chain variabledomains comprise three complementarity determining regions (CDRs) whichdirect binding to the same antigen; (2) cultivating said host cell underconditions such that said heavy chain variable and constant domains andsaid light chain variable and constant domains are produced; and (3)recovering from said host cell a Fab comprising said heavy chainvariable and constant domains and said light chain variable and constantdomains. More particular to this embodiment, the present inventionprovides a method comprising the following: said first nucleic acidencodes a heavy chain variable domain further comprising an argininesubstituted at residue 105 (105R) and said second nucleic acid encodes alight chain variable domain further comprising an aspartic acidsubstituted at residue 42 (42D).

In a separate embodiment, the present invention provides a method forproducing a fragment, antigen binding (Fab) comprising: (1)co-expressing in a host cell: (a) a first nucleic acid encoding both aheavy chain variable domain and an IgG heavy chain constant CH1 domain,wherein said heavy chain variable domain comprises a tyrosinesubstituted at residue 39 (39Y) and said heavy chain CH1 domaincomprises an aspartic acid substituted at residue 228 (228D); and (b) asecond nucleic acid encoding both a light chain variable domain and alight chain constant domain wherein said light chain variable domaincomprises an arginine substituted at residue 38 (38R) and said lightchain constant domain comprises a lysine substituted at residue 122(122K), wherein each of said heavy chain and light chain variabledomains comprise three complementarity determining regions (CDRs) whichdirect binding to the same antigen; (2) cultivating said host cell underconditions such that said heavy chain variable and constant domains andsaid light chain variable and constant domains are produced; and (3)recovering from said host cell a Fab comprising said heavy chainvariable and constant domains and said light chain variable and constantdomains. More particular to this embodiment, the present inventionprovides a method comprising the following: said first nucleic acidencodes a heavy chain variable domain further comprising an argininesubstituted at residue 105 (105R) and said second nucleic acid encodes alight chain variable domain further comprising an aspartic acidsubstituted at residue 42 (42D).

The present invention also provides a method for producing a fragment,antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a)a first nucleic acid encoding both a heavy chain variable domain and anIgG heavy chain constant CH1 domain, wherein said heavy chain variabledomain comprises a lysine substituted at residue 39 (39K) and a glutamicacid substituted at the residue which is four amino acids upstream ofthe first residue of HFR3 according to Kabat, and said heavy chain CH1domain comprises a WT sequence; and (b) a second nucleic acid encodingboth a light chain variable domain and a light chain constant domainwherein said light chain variable domain is a kappa isotype andcomprises an arginine substituted at residue 1 (1R) and an aspartic acidsubstituted at residue 38 (38D), and said light chain constant domaincomprises a WT sequence, wherein each of said heavy chain and lightchain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to the same antigen; (2) cultivatingsaid host cell under conditions such that said heavy chain variable andconstant domains and said light chain variable and constant domains areproduced; and (3) recovering from said host cell a Fab comprising saidheavy chain variable and constant domains and said light chain variableand constant domains.

More particularly, the present invention provides a method for producinga fragment, antigen binding (Fab) comprising: (1) co-expressing in ahost cell: (a) a first nucleic acid encoding both a heavy chain variabledomain and an IgG heavy chain constant CH1 domain, wherein said heavychain variable domain comprises a lysine substituted at residue 39 (39K)and a glutamic acid substituted at the residue which is four amino acidsupstream of the first residue of HFR3 according to Kabat, and said IgGheavy chain constant CH1 domain comprises an aspartic acid substitutedat residue 228 (228D); and (b) a second nucleic acid encoding both alight chain variable domain and a light chain constant domain whereinsaid light chain variable domain is a kappa isotype and comprises anarginine substituted at residue 1 (1R) and an aspartic acid substitutedat residue 38 (38D) and said light chain constant domain comprises alysine substituted at residue 122 (122K), wherein each of said heavychain and light chain variable domains comprise three complementaritydetermining regions (CDRs) which direct binding to the same antigen; (2)cultivating said host cell under conditions such that said heavy chainvariable and constant domains and said light chain variable and constantdomains are produced; and (3) recovering from said host cell a Fabcomprising said heavy chain variable and constant domains and said lightchain variable and constant domains.

The present invention also provides a method for producing a fragment,antigen binding (Fab) comprising: (1) co-expressing in a host cell: (a)a first nucleic acid encoding both a heavy chain variable domain and anIgG heavy chain constant CH1 domain, wherein said heavy chain variabledomain comprises a tyrosine substituted at residue 39 (39Y) and saidheavy chain CH1 domain comprises an alanine substituted at residue 172(172A) and a glycine substituted at residue 174 (174G); and (b) a secondnucleic acid encoding both a light chain variable domain and a lightchain constant domain wherein said light chain variable domain comprisesan arginine substituted at residue 38 (38R) and said light chainconstant domain comprises a tyrosine substituted at residue 135 (135Y)and a tryptophan substituted at residue 176 (176W), wherein each of saidheavy chain and light chain variable domains comprise threecomplementarity determining regions (CDRs) which direct binding to thesame antigen; (2) cultivating said host cell under conditions such thatsaid heavy chain variable and constant domains and said light chainvariable and constant domains are produced; and (3) recovering from saidhost cell a Fab comprising said heavy chain variable and constantdomains and said light chain variable and constant domains. Moreparticular to this embodiment, the present invention provides a methodcomprising the following: said first nucleic acid encodes a heavy chainvariable domain further comprising an arginine substituted at residue105 (105R) and said second nucleic acid encodes a light chain variabledomain further comprising an aspartic acid substituted at residue 42(42D).

In a more particular embodiment, the present invention provides a methodfor producing a first and second fragment, antigen binding (Fab)comprising: (1) co-expressing in a host cell: (a) a first nucleic acidencoding both a first heavy chain variable domain and a first IgG heavychain constant CH1 domain, wherein said first heavy chain variabledomain comprises a lysine substituted at residue 39 (39K) and a glutamicacid substituted at the residue which is four amino acids upstream ofthe first residue of HFR3 according to Kabat, and said first IgG heavychain constant CH1 domain comprises an alanine substituted at residue172 (172A) and a glycine substituted at residue 174 (174G); (b) a secondnucleic acid encoding both a first light chain variable domain and afirst light chain constant domain, wherein said first light chainvariable domain is a kappa isotype and comprises an arginine substitutedat residue 1(1R) and an aspartic acid substituted at residue 38 (38D),and said first light chain constant domain comprises a tyrosinesubstituted at residue 135 (135Y) and a tryptophan substituted atresidue 176 (176W); (c) a third nucleic acid encoding both a secondheavy chain variable domain and a second IgG heavy chain constant CH1domain, wherein said second heavy chain variable domain comprises atyrosine substituted at residue 39 (39Y) and said second IgG heavy chainconstant CH1 domain comprises a WT sequence; and (d) a fourth nucleicacid encoding both a second light chain variable domain and a secondlight chain constant domain, wherein said second light chain variabledomain comprises an arginine substituted at residue 38 (38R) and saidsecond light chain constant domain comprises a WT sequence, wherein eachof said first heavy chain and light chain variable domains comprisethree complementarity determining regions (CDRs) which direct binding toa first antigen and further wherein each of said second heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a second antigen that differsfrom said first antigen; (2) cultivating said host cell under conditionssuch that said first and second heavy chain variable and IgG CH1constant domains and said first and second light chain variable andconstant domains are produced; and (3) recovering from said host cell afirst and second Fab wherein said first Fab comprises said first heavychain variable and constant domains and said first light chain variableand constant domains, and said second Fab comprises said second heavychain variable and constant domains and said second light chain variableand constant domains. More particular to this embodiment, the presentinvention provides a method comprising one or more of the following:said first nucleic acid encodes a heavy chain CH1 constant domainfurther comprising a methionine or isoleucine substituted at residue 190(190M or 190I); said second nucleic acid encodes a light chain constantdomain further comprising an leucine substituted at residue 133 (133L);said second nucleic acid encodes a light chain constant domain furthercomprising a glutamine or aspartic acid substituted at residue 174 (174Qor 174D), and said third nucleic acid encodes a heavy chain variabledomain further comprising an arginine substituted at residue 105 (105R)with said fourth nucleic acid encoding a light chain variable domainfurther comprising an aspartic acid substituted at residue 42 (42D).

In a further embodiment, the present invention provides a method forproducing a first and second fragment, antigen binding (Fab) comprising:(1) co-expressing in a host cell: (a) a first nucleic acid encoding botha first heavy chain variable domain and a first IgG heavy chain constantCH1 domain, wherein said first heavy chain variable domain comprises alysine substituted at residue 39 (39K) and a glutamic acid substitutedat the residue which is four amino acids upstream of the first residueof HFR3 according to Kabat, and said first IgG heavy chain constant CH1domain comprises an arginine substituted at residue 172 (172R) and aglycine substituted at residue 174 (174G); (b) a second nucleic acidencoding both a first light chain variable domain and a first lightchain constant domain, wherein said first light chain variable domain isa kappa isotype and comprises an arginine substituted at residue 1 (1R)and, an aspartic acid substituted at residue 38 (38D), and said firstlight chain constant domain comprises a tyrosine substituted at residue135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) athird nucleic acid encoding both a second heavy chain variable domainand a second IgG heavy chain constant CH1 domain, wherein said secondheavy chain variable domain comprises a tyrosine substituted at residue39 (39Y) and said second IgG heavy chain constant CH1 domain comprises aWT sequence; and (d) a fourth nucleic acid encoding both a second lightchain variable domain and a second light chain constant domain, whereinsaid second light chain variable domain comprises an argininesubstituted at residue 38 (38R) and said second light chain constantdomain comprises a WT sequence, wherein each of said first heavy chainand light chain variable domains comprise three complementaritydetermining regions (CDRs) which direct binding to a first antigen andfurther wherein each of said second heavy chain and light chain variabledomains comprise three complementarity determining regions (CDRs) whichdirect binding to a second antigen that differs from said first antigen;(2) cultivating said host cell under conditions such that said first andsecond heavy chain variable and IgG CH1 constant domains and said firstand second light chain variable and constant domains are produced; and(3) recovering from said host cell a first and second Fab wherein saidfirst Fab comprises said first heavy chain variable and constant domainsand said first light chain variable and constant domains, and saidsecond Fab comprises said second heavy chain variable and constantdomains and said second light chain variable and constant domains. Moreparticular to this embodiment, the present invention provides a methodcomprising one or more of the following: said first nucleic acid encodesa heavy chain all constant domain further comprising a methionine orisoleucine substituted at residue 190 (190M or 190I); said secondnucleic acid encodes a light chain constant domain further comprising aleucine substituted at residue 133 (133L); said second nucleic acidencodes a light chain constant domain further comprising a glutamine oraspartic acid substituted at residue 174 (174Q or 174D), and said thirdnucleic acid encodes a heavy chain variable domain further comprising anarginine substituted at residue 105 (105R) with said fourth nucleic acidencoding a light chain variable domain further comprising an asparticacid substituted at residue 42 (42D).

In a separate embodiment, the present invention provides A method forproducing a first and second fragment, antigen binding (Fab) comprising:(1) co-expressing in a host cell: (a) a first nucleic acid encoding botha first heavy chain variable domain and a first IgG heavy chain constantCH1 domain, wherein said first heavy chain variable domain comprises alysine substituted at residue 39 (39K) and a glutamic acid substitutedat the residue which is four amino acids upstream of the first residueof HFR3 according to Kabat, and said first IgG heavy chain constant CH1domain comprises an alanine substituted at residue 172 (172A) and aglycine substituted at residue 174 (174G); (b) a second nucleic acidencoding both a first light chain variable domain and a first lightchain constant domain, wherein said first light chain variable domain isa kappa isotype and comprises an arginine substituted at residue 1 (1R)and an aspartic acid substituted at residue 38 (38D), and said firstlight chain constant domain comprises a tyrosine substituted at residue135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) athird nucleic acid encoding both a second heavy chain variable domainand a second IgG heavy chain constant CH1 domain, wherein said secondheavy chain variable domain comprises a tyrosine substituted at residue39 (39Y) and said second IgG heavy chain constant CH1 domain comprisesan aspartic acid substituted at residue 228 (228D); and (d) a fourthnucleic acid encoding both a second light chain variable domain and asecond light chain constant domain, wherein said second light chainvariable domain comprises an arginine substituted at residue 38 (38R)and said second light chain constant domain comprises a lysinesubstituted at residue 122 (122K), wherein each of said first heavychain and light chain variable domains comprise three complementaritydetermining regions (CDRs) which direct binding to a first antigen andfurther wherein each of said second heavy chain and light chain variabledomains comprise three complementarity determining regions (CDRs) whichdirect binding to a second antigen that differs from said first antigen;(2) cultivating said host cell under conditions such that said first andsecond heavy chain variable and IgG CH1 constant domains and said firstand second light chain variable and constant domains are produced; and(3) recovering from said host cell a first and second Fab wherein saidfirst Fab comprises said first heavy chain variable and constant domainsand said first light chain variable and constant domains, and saidsecond Fab comprises said second heavy chain variable and constantdomains and said second light chain variable and constant domains. Moreparticular to this embodiment, the present invention provides a methodcomprising one or more of the following: said first nucleic acid encodesa heavy chain CH1 constant domain further comprising a methionine orisoleucine substituted at residue 190 (190M or 190I); said secondnucleic acid encodes a light chain constant domain further comprising aleucine substituted at residue 133 (133L); said second nucleic acidencodes a light chain constant domain further comprising a glutamine oraspartic acid substituted at residue 174 (174Q or 174D), and said thirdnucleic acid encodes a heavy chain variable domain further comprising anarginic substituted at residue 105 (105R) with said fourth nucleic acidencoding a light chain variable domain further comprising an asparticacid substituted at residue 42 (42D).

In another embodiment, the present invention provides a method forproducing a first and second fragment, antigen binding (Fab) comprising:(1) co-expressing in a host cell: (a) a first nucleic acid encoding botha first heavy chain variable domain and a first IgG heavy chain constantCH1 domain, wherein said first heavy chain variable domain comprises alysine substituted at residue 39 (39K) and a glutamic acid substitutedat the residue which is four amino acids upstream of the first residueof HFR3 according to Kabat, and said first IgG heavy chain constant CH1domain comprises an arginine substituted at residue 172 (172R) and aglycine substituted at residue 174 (174G); (b) a second nucleic acidencoding both a first light chain variable domain and a first lightchain constant domain, wherein said first light chain variable domain isa kappa isotype and comprises an arginine substituted at residue 1 (1R)and an aspartic acid substituted at residue 38 (38D), and said firstlight chain constant domain comprises a tyrosine substituted at residue135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) athird nucleic acid encoding both a second heavy chain variable domainand a second IgG heavy chain constant CH1 domain, wherein said secondheavy chain variable domain comprises a tyrosine substituted al residue39 (39Y) and said second IgG heavy chain constant CH1 domain comprisesan aspartic acid substituted at residue 228 (228D); and (d) a fourthnucleic acid encoding both a second light chain variable domain and asecond light chain constant domain, wherein said second light chainvariable domain comprises an arginine substituted at residue 38 (38R)and said second light chain constant domain comprises a lysinesubstituted at residue 122 (122K), wherein each of said first heavychain and light chain variable domains comprise three complementaritydetermining regions (CDRs) which direct binding to a first antigen andfurther wherein each of said second heavy chain and light chain variabledomains comprise three complementarity determining regions (CDRs) whichdirect binding to a second antigen that differs from said first antigen;(2) cultivating said host cell under conditions such that said first andsecond heavy chain variable and IgG CH1 constant domains and said firstand second light chain variable and constant domains are produced; and(3) recovering from said host cell a first and second Fab wherein saidfirst Fab comprises said first heavy chain variable and constant domainsand said first light chain variable and constant domains, and saidsecond Fab comprises said second heavy chain variable and constantdomains and said second light chain variable and constant domains. Moreparticular to this embodiment, the present invention provides a methodcomprising one or more of the following: said first nucleic acid encodesa heavy chain CH1 constant domain further comprising a methionine orisoleucine substituted at residue 190 (190M or 190I); said secondnucleic acid encodes a light chain constant domain further comprising aleucine substituted at residue 133 (133L); said second nucleic acidencodes a light chain constant domain further comprising a glutamine oraspartic acid substituted at residue 174 (174Q or 174D), and said thirdnucleic acid encodes a heavy chain variable domain further comprising anarginic substituted at residue 105 (105R) with said fourth nucleic acidencoding a light chain variable domain further comprising an asparticacid substituted at residue 42 (42D).

In another particular embodiment, the present invention provides amethod for producing a first and second fragment, antigen binding (Fab)comprising: (1) co-expressing in a host cell: (a) a first nucleic acidencoding both a first heavy chain variable domain and a first IgG heavychain constant CH1 domain, wherein said first heavy chain variabledomain comprises a lysine substituted at residue 39 (39K) and a glutamicacid substituted at the residue which is four amino acids upstream ofthe first residue of HFR3 according to Kabat, and said first IgG heavychain constant CH1 domain comprises an alanine substituted at residue172 (172A) and a glycine substituted at residue 174 (174G); (b) a secondnucleic acid encoding both a first light chain variable domain and afirst light chain constant domain, wherein said first light chainvariable domain is a kappa isotype and comprises an arginine substitutedat residue 1(1R) and an aspartic acid substituted at residue 38 (38D),and said first light chain constant domain comprises a phenylalaninesubstituted at residue 135 (135F) and a tryptophan substituted atresidue 176 (176W); (c) a third nucleic acid encoding both a secondheavy chain variable domain and a second IgG heavy chain constant CH1domain, wherein said second heavy chain variable domain comprises atyrosine substituted at residue 39 (39Y) and said second IgG heavy chainconstant CH1 domain comprises a WT sequence; and (d) a fourth nucleicacid encoding both a second light chain variable domain and a secondlight chain constant domain, wherein said second light chain variabledomain comprises an arginine substituted at residue 38 (38R) and saidsecond light chain constant domain comprises a WT sequence, wherein eachof said first heavy chain and light chain variable domains comprisethree complementarity determining regions (CDRs) which direct binding toa first antigen and further wherein each of said second heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a second antigen that differsfrom said first antigen; (2) cultivating said host cell under conditionssuch that said first and second heavy chain variable and IgG CH1constant domains and said first and second light chain variable andconstant domains are produced; and (3) recovering from said host cell afirst and second Fab wherein said first Fab comprises said first heavychain variable and constant domains and said first light chain variableand constant domains, and said second Fab comprises said second heavychain variable and constant domains and said second light chain variableand constant domains.

The present invention also provides a method for producing a first andsecond fragment, antigen binding (Fab) comprising: (1) co-expressing ina host cell: (a) a first nucleic acid encoding both a first heavy chainvariable domain and a first IgG heavy chain constant CH1 domain, whereinsaid first heavy chain variable domain comprises a tyrosine substitutedat residue 39 (39Y), and said first IgG heavy chain constant all domaincomprises an alanine substituted at residue 172 (172A) and a glycinesubstituted at residue 174 (174G); (b) a second nucleic acid encodingboth a first light chain variable domain and a first light chainconstant domain, wherein said first light chain variable domaincomprises an arginine substituted at residue 38 (38R), and said firstlight chain constant domain comprises a tyrosine substituted at residue135 (135Y) and a tryptophan substituted at residue 176 (176W); (c) athird nucleic acid encoding both a second heavy chain variable domainand a second IgG heavy chain constant CH1 domain, wherein said secondheavy chain variable domain comprises a lysine substituted at residue 39(39K) and a glutamic acid substituted at the residue which is four aminoacids upstream of the first residue of HFR3 according to Kabat and saidsecond IgG heavy chain constant CH1 domain comprises a WT sequence; and(d) a fourth nucleic acid encoding both a second light chain variabledomain and a second light chain constant domain, wherein said lightchain variable domain is a kappa isotype and comprises an argininesubstituted at residue 1 (1R) and an aspartic acid substituted atresidue 38 (38D) and said second light chain constant domain comprises aWT sequence, wherein each of said first heavy chain and light chainvariable domains comprise three complementarity determining regions(CDRs) which direct binding to a first antigen and further wherein eachof said second heavy chain and light chain variable domains comprisethree complementarity determining regions (CDRs) which direct binding toa second antigen that differs from said first antigen; (2) cultivatingsaid host cell under conditions such that said first and second heavychain variable and IgG CH1 constant domains and said first and secondlight chain variable and constant domains are produced; and (3)recovering from said host cell a first and second Fab wherein said firstFab comprises said first heavy chain variable and constant domains andsaid first light chain variable and constant domains, and said secondFab comprises said second heavy chain variable and constant domains andsaid second light chain variable and constant domains. More particularto this embodiment, the present invention provides a method comprisingthe following: said first nucleic acid encodes a heavy chain variabledomain further comprising an arginine substituted at residue 105 (105R)and said second nucleic acid encodes a light chain variable domainfurther comprising an aspartic acid substituted at residue 42 (42D).

In another embodiment, the present invention provides a method forproducing a first and second fragment, antigen binding (Fab) comprising:(1) co-expressing in a host cell: (a) a first nucleic acid encoding botha first heavy chain variable domain and a first IgG heavy chain constantCH1 domain, wherein said first heavy chain variable domain comprises atyrosine substituted at residue 39 (39Y), and said first IgG heavy chainconstant CH1 domain comprises an alanine substituted at residue 172(172A) and a glycine substituted at residue 174 (174G); (b) a secondnucleic acid encoding both a first light chain variable domain and afirst light chain constant domain, wherein said first light chainvariable domain comprises an arginine substituted at residue 38 (38R),and said first light chain constant domain comprises a tyrosinesubstituted at residue 135 (135Y) and a tryptophan substituted atresidue 176 (176W); (c) a third nucleic acid encoding both a secondheavy chain variable domain and a second IgG heavy chain constant CH1domain, wherein said second heavy chain variable domain comprises alysine substituted at residue 39 (39K) and a glutamic acid substitutedat the residue which is four amino acids upstream of the first residueof HFR3 according to Kabat and said second IgG heavy chain constant CH1domain comprises an aspartic acid substituted at residue 228 (228D); and(d) a fourth nucleic acid encoding both a second light chain variabledomain and a second light chain constant domain, wherein said lightchain variable domain is a kappa isotype and comprises an argininesubstituted at residue 1 (1R) and an aspartic acid substituted atresidue 38 (38D) and said second light chain constant domain comprises alysine substituted at residue 122 (122K), wherein each of said firstheavy chain and light chain variable domains comprise threecomplementarity determining regions (CDRs) which direct binding to afirst antigen and further wherein each of said second heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a second antigen that differsfrom said first antigen; (2) cultivating said host cell under conditionssuch that said first and second heavy chain variable and IgG CH1constant domains and said first and second light chain variable andconstant domains are produced; and (3) recovering from said host cell afirst and second Fab wherein said first Fab comprises said first heavychain variable and constant domains and said first light chain variableand constant domains, and said second Fab comprises said second heavychain variable and constant domains and said second light chain variableand constant domains. More particular to this embodiment, the presentinvention provides a method comprising the following; said first nucleicacid encodes a heavy chain variable domain further comprising anarginine substituted at residue 105 (105R) and said second nucleic acidencodes a light chain variable domain further comprising an asparticacid substituted at residue 42 (42D).

The present invention also provides a method for producing a bispecificantibody comprising: (1) co-expressing in a host cell: (a) a firstnucleic acid encoding a first IgG heavy chain, wherein said first heavychain comprises a variable domain comprising a lysine substituted atresidue 39 (39K) and a glutamic acid substituted at the residue which isfour amino acids upstream of the first residue of HFR3 according toKabat, and a CH1 constant domain comprising an alanine substituted atresidue 172 (172A) and a glycine substituted at residue 174 (174G); (b)a second nucleic acid encoding a first light chain, wherein said firstlight chain comprises a kappa variable domain comprising an argininesubstituted at residue 1(1R) and an aspartic acid substituted at residue38 (38D), and a constant domain comprising a tyrosine substituted atresidue 135 (135Y) and a tryptophan substituted at residue 176 (176W);(c) a third nucleic acid encoding a second IgG heavy chain, wherein saidsecond heavy chain comprises a variable domain comprising a tyrosinesubstituted at residue 39 (39Y), and a CH1 constant domain comprising aWT sequence; and (d) a fourth nucleic acid encoding a second lightchain, wherein said second light chain comprises a variable domaincomprising an arginine substituted at residue 38 (38R) and a constantdomain comprising a WT sequence, wherein each of said first heavy chainand light chain variable domains comprise three complementaritydetermining regions (CDRs) which direct binding to a first antigen andfurther wherein each of said second heavy chain and light chain variabledomains comprise three complementarity determining regions (CDRs) whichdirect binding to a second antigen that differs from said first antigen;(2) cultivating said host cell under conditions such that said first andsecond IgG heavy chains and said first and second light chains areproduced; and (3) recovering from said host cell a bispecific antibodycomprising a first and second fragment, antigen binding (Fab) whereinsaid first Fab comprises said first IgG heavy chain and said first lightchain and said second Fab comprises said second IgG heavy chain and saidsecond light chain. More particular to this embodiment, the presentinvention provides a method comprising one or more of the following:said first nucleic acid encodes a heavy chain CH1 constant domainfurther comprising a methionine or isoleucine substituted at residue 190(190M or 190I); said second nucleic acid encodes a light chain constantdomain further comprising a leucine substituted at residue 133 (133L);said second nucleic acid encodes a light chain constant domain furthercomprising a glutamine or aspartic acid substituted at residue 174 (174Qor 174D), and said third nucleic acid encodes a heavy chain variabledomain further comprising an arginine substituted at residue 105 (105R)with said fourth nucleic acid encoding a light chain variable domainfurther comprising an aspartic acid substituted at residue 42 (42D).

In a separate embodiment, the present invention provides a method forproducing a bispecific antibody comprising: (1) co-expressing in a hostcell: (a) a first nucleic acid encoding a first IgG heavy chain, whereinsaid first heavy chain comprises a variable domain comprising a lysinesubstituted at residue 39 (39K) and a glutamic acid substituted at theresidue which is 4 amino acids upstream of the first residue of HFR3according to Kabat, and a CH1 constant domain comprising an argininesubstituted at residue 172 (172R) and a glycine substituted at residue174 (174G); (b) a second nucleic acid encoding a first light chain,wherein said first light chain comprises a kappa variable domaincomprising an arginine substituted at residue 1 (1R) and an asparticacid substituted at residue 38 (38D), and a constant domain comprising atyrosine substituted at residue 135 (135Y) and a tryptophan substitutedat residue 176 (176W); (c) a third nucleic acid encoding a second IgGheavy chain, wherein said second heavy chain comprises a variable domaincomprising a tyrosine substituted at residue 39 (39Y), and a CH1constant domain comprising a WT sequence; and (d) a fourth nucleic acidencoding a second light chain, wherein said second light chain comprisesa variable domain comprising an arginine substituted at residue 38 (38R)and a constant domain comprising a WT sequence, wherein each of saidfirst heavy chain and light chain variable domains comprise threecomplementarity determining regions (CDRs) which direct binding to afirst antigen and further wherein each of said second heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a second antigen that differsfrom said first antigen; (2) cultivating said host cell under conditionssuch that said first and second IgG heavy chains and said first andsecond light chains are produced; and (3) recovering from said host cella bispecific antibody comprising a first and second fragment, antigenbinding (Fab) wherein said first Fab comprises said first IgG heavychain and said first light chain and said second Fab comprises saidsecond IgG heavy chain and said second light chain. More particular tothis embodiment, the present invention provides a method comprising oneor more of the following: said first nucleic acid encodes a heavy chainCH1 constant domain further comprising a methionine or isoleucinesubstituted at residue 190 (190M or 190I); said second nucleic acidencodes a light chain constant domain further comprising a leucinesubstituted at residue 133 (133L); said second nucleic acid encodes alight chain constant domain further comprising a glutamine or asparticacid substituted at residue 174 (174Q or 174D), and said third nucleicacid encodes a heavy chain variable domain further comprising anarginine substituted at residue 105 (105R) with said fourth nucleic acidencoding a light chain variable domain further comprising an asparticacid substituted at residue 42 (42D).

In yet another embodiment, the present invention provides a method forproducing a bispecific antibody comprising: (1) co-expressing in a hostcell: (a) a first nucleic acid encoding a first IgG heavy chain, whereinsaid first heavy chain comprises a variable domain comprising a lysinesubstituted at residue 39 (39K) and a glutamic acid substituted at theresidue which is four amino acids upstream of the first residue of HFR3according to Kabat, and a CH1 constant domain comprising an alaninesubstituted at residue 172 (172A) and a glycine substituted at residue174 (174G); (b) a second nucleic acid encoding a first light chain,wherein said first light chain comprises a kappa variable domaincomprising an arginine substituted at residue 1 (1R) and an asparticacid substituted at residue 38 (38D), and a constant domain comprising atyrosine substituted at residue 135 (135Y) and a tryptophan substitutedat residue 176 (176W); (c) a third nucleic acid encoding a second IgGheavy chain, wherein said second heavy chain comprises a variable domaincomprising a tyrosine substituted at residue 39 (39Y), and a CH1constant domain comprising an aspartic acid substituted at residue 228(228D); and (d) a fourth nucleic acid encoding a second light chain,wherein said second light chain comprises a variable domain comprisingan arginine substituted at residue 38 (38R) and a constant domaincomprising a lysine substituted at residue 122 (122K), wherein each ofsaid first heavy chain and light chain variable domains comprise threecomplementarity determining regions (CDRs) which direct binding to afirst antigen and further wherein each of said second heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a second antigen that differsfrom said first antigen; (2) cultivating said host cell under conditionssuch that said first and second IgG heavy chains and said first andsecond light chains are produced; and (3) recovering from said host cella bispecific antibody comprising a first and second fragment, antigenbinding (Fab) wherein said first Fab comprises said first IgG heavychain and said first light chain and said second Fab comprises saidsecond IgG heavy chain and said second light chain. More particular tothis embodiment, the present invention provides a method comprising oneor more of the following: said first nucleic acid encodes a heavy chainCH1 constant domain further comprising a methionine or isoleucinesubstituted at residue 190 (190M or 190I); said second nucleic acidencodes a light chain constant domain further comprising a leucinesubstituted at residue 133 (133L); said second nucleic acid encodes alight chain constant domain further comprising a glutamine or asparticacid substituted at residue 174 (174Q or 174D), and said third nucleicacid encodes a heavy chain variable domain further comprising anarginine substituted at residue 105 (105R) with said fourth nucleic acidencoding a light chain variable domain further comprising an asparticacid substituted at residue 42 (42D).

In another embodiment, the present invention provides a method forproducing a bispecific antibody comprising: (1) co-expressing in a hostcell: (a) a first nucleic acid encoding a first IgG heavy chain, whereinsaid first heavy chain comprises a variable domain comprising a lysinesubstituted at residue 39 (39K) and a glutamic acid substituted at theresidue which is four amino acids upstream of the first residue of HFR3according to Kabat, and a CH1 constant domain comprising an argininesubstituted at residue 172 (172R) and a glycine substituted at residue174 (174G); (b) a second nucleic acid encoding a first light chain,wherein said first light chain comprises a kappa variable domaincomprising an arginine substituted at residue 1 (1R) and an asparticacid substituted at residue 38 (38D), and a constant domain comprising atyrosine substituted at residue 135 (135Y) and a tryptophan substitutedat residue 176 (176W); (c) a third nucleic acid encoding a second IgGheavy chain, wherein said second heavy chain comprises a variable domaincomprising a tyrosine substituted at residue 39 (39Y), and a CH1constant domain comprising an aspartic acid substituted at residue 228(228D); and (d) a fourth nucleic acid encoding a second light chain,wherein said second light chain comprises a variable domain comprisingan arginine substituted at residue 38 (38R) and a constant domaincomprising a lysine substituted at residue 122 (122K), wherein each ofsaid first heavy chain and light chain variable domains comprise threecomplementarity determining regions (CDRs) which direct binding to afirst antigen and further wherein each of said second heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a second antigen that differsfrom said first antigen; (2) cultivating said host cell under conditionssuch that said first and second IgG heavy chains and said first andsecond light chains are produced; and (3) recovering from said host cella bispecific antibody comprising a first and second fragment, antigenbinding (Fab) wherein said first Fab comprises said first IgG heavychain and said first light chain and said second Fab comprises saidsecond IgG heavy chain and said second light chain. More particular tothis embodiment, the present invention provides a method comprising oneor more of the following: said first nucleic acid encodes a heavy chainCH1 constant domain further comprising a methionine or isoleucinesubstituted at residue 190 (190M or 190I); said second nucleic acidencodes a light chain constant domain further comprising a leucinesubstituted at residue 133 (133L); said second nucleic acid encodes alight chain constant domain further comprising a glutamine or asparticacid substituted at residue 174 (174Q or 174D), and said third nucleicacid encodes a heavy chain variable domain further comprising anarginine substituted at residue 105 (105R) with said fourth nucleic acidencoding a light chain variable domain further comprising an asparticacid substituted at residue 42 (42D).

The present invention also provides a method for producing a bispecificantibody comprising: (1) co-expressing in a host cell: (a) a firstnucleic acid encoding a first IgG heavy chain, wherein said first heavychain comprises a variable domain comprising a tyrosine substituted atresidue 39 (39Y), and a CH1 constant domain comprising an alaninesubstituted at residue 172 (172A) and a glycine substituted at residue174 (174G); (b) a second nucleic acid encoding a first light chain,wherein said first light chain comprises a variable domain comprising anarginine substituted at residue 38 (38R), and a constant domaincomprising a tyrosine substituted at residue 135 (135Y) and a tryptophansubstituted at residue 176 (176W); (c) a third nucleic acid encoding asecond IgG heavy chain, wherein said heavy chain comprises a variabledomain comprising a lysine substituted at residue 39 (39K) and aglutamic acid substituted at the residue which is four amino acidsupstream of the first residue of HFR3 according to Kabat and a constantdomain comprising a WT sequence: and (d) a fourth nucleic acid encodingsecond light chain, wherein said second light chain comprises a kappavariable domain comprising an arginine substituted at residue 1 (1R) andan aspartic acid substituted at residue 38 (38D), and a constant domaincomprising a WT sequence, wherein each of said first heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a first antigen and furtherwherein each of said second heavy chain and light chain variable domainscomprise three complementarity determining regions (CDRs) which directbinding to a second antigen that differs from said first antigen; (2)cultivating said host cell under conditions such that said first andsecond IgG heavy chains and said first and second light chains areproduced; and (3) recovering from said host cell a bispecific antibodycomprising a first and second fragment, antigen binding (Fab) whereinsaid first Fab comprises said first IgG heavy chain and said first lightchain and said second Fab comprises said second IgG heavy chain and saidsecond light chain. More particular to this embodiment, the presentinvention provides a method comprising the following: said first nucleicacid encodes a heavy chain variable domain further comprising anarginine substituted at residue 105 (105R) and said second nucleic acidencodes a light chain variable domain further comprising an asparticacid substituted at residue 42 (42D).

The present invention also provides a method for producing a bispecificantibody comprising: (1) co-expressing in a host cell: (a) a firstnucleic acid encoding a first IgG heavy chain, wherein said, first heavychain comprises a variable domain comprising a tyrosine substituted atresidue 39(39Y), and a CH1 constant domain comprising an alaninesubstituted at residue 172 (172A) and a glycine substituted at residue174 (174G); (b) a second nucleic acid encoding a first light chain,wherein said first light chain comprises a variable domain comprising anarginine substituted at residue 38 (38R), and a constant domaincomprising a tyrosine substituted at residue 135 (135Y) and a tryptophansubstituted at residue 176 (I76W); (c) a third nucleic acid encoding asecond IgG heavy chain, wherein said heavy chain comprises a variabledomain comprising a lysine substituted at residue 39 (39K) and aglutamic acid substituted at the residue which is four amino acidsupstream of the first residue of HFR3 according to Kabat and a constantdomain comprising an aspartic acid substituted at residue 228 (228D);and (d) a fourth nucleic acid encoding second light chain, wherein saidsecond light chain comprises a kappa variable domain comprising anarginine substituted at residue 1 (1R) and an aspartic acid substitutedat residue 38 (38D), and a constant domain comprising a lysinesubstituted at residue 122 (122K), wherein each of said first heavychain and light chain variable domains comprise three complementaritydetermining regions (CDRs) which direct binding to a first antigen andfurther wherein each of said second heavy chain and light chain variabledomain's comprise three complementarity determining regions (CDRs) whichdirect binding to a second antigen that differs from said first antigen;(2) cultivating said host cell under conditions such that said first andsecond IgG heavy chains and said first and second light chains areproduced; and (3) recovering from said host cell a bispecific antibodycomprising a first and second fragment, antigen binding (Fab) whereinsaid first Fab comprises said first IgG heavy chain and said first lightchain and said second Fab comprises said second IgG heavy chain and saidsecond light chain. More particular to this embodiment, the presentinvention provides a method comprising the following: said first nucleicacid encodes a heavy chain variable domain further comprising anarginine substituted at residue 105 (105R) and said second nucleic acidencodes a light chain variable domain further comprising an asparticacid substituted at residue 42 (42D).

As a further particular embodiment to the methods for producing abispecific antibody, as provided herein, the present invention providesa method wherein one of said first and second IgG heavy chains furthercomprises a CH3 constant domain comprising a lysine substituted atresidue 356 and a lysine substituted at residue 399, and the other ofsaid first and second IgG heavy chains further comprises a CH3 constantdomain comprising an aspartic acid substituted at residue 392 and anaspartic acid substituted at residue 409.

Even more particularly, the present invention provides a method forproducing a bispecific antibody comprising: (1) co-expressing in a hostcell: (a) a first nucleic acid encoding a first IgG heavy chain, whereinsaid first heavy chain comprises a variable domain comprising a lysinesubstituted at residue 39 (39K) and a glutamic acid substituted at theresidue which is four amino acids upstream of the first residue of HFR3according to Kabat, a CH1 constant domain comprising an alaninesubstituted at residue 172 (172A) and a glycine substituted at residue174 (174G) and a CH3 constant domain comprising a lysine substituted atresidue 356 (356K) and a lysine substituted at residue 399 (399K); (b) asecond nucleic acid encoding a first light chain, wherein said firstlight chain comprises a kappa variable domain comprising an argininesubstituted at residue 1 (1R) and an aspartic acid substituted atresidue 38 (38D), and a constant domain comprising a tyrosinesubstituted at residue 135 (135Y) and a tryptophan substituted atresidue 176 (176W); (c) a third nucleic acid encoding a second IgG heavychain, wherein said second heavy chain comprises a variable domaincomprising a tyrosine substituted at residue 39 (39Y), a CH1 constantdomain comprising a WT sequence and a CH3 constant domain comprising anaspartic acid substituted at residue 392 (392D) and an aspartic acidsubstituted at residue 409 (409D); and (d) a fourth nucleic acidencoding a second light chain, wherein said second light chaincomprises, a variable domain comprising an arginine substituted atresidue 38 (38R) and a constant domain comprising a WT sequence, whereineach of said first heavy chain and light chain variable domains comprisethree complementarity determining regions (CDRs) which direct binding toa first antigen and further wherein each of said second heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a second antigen that differsfrom said first antigen; (2) cultivating said host cell under conditionssuch that said first and second IgG heavy chains and said first andsecond light chains are produced; and (3) recovering from said host cella bispecific antibody comprising a first and second fragment, antigenbinding (Fab) wherein said first Fab comprises said first IgG heavychain and said first light chain and said second Fab comprises saidsecond IgG heavy chain and said second light chain.

In other particular embodiments, the host cell for use in the methods ofthe present invention is a mammalian cell, more particularly a HEK293 orCHO cell, and the IgG heavy chain constant domain produced by themethods of the present invention is IgG1 or IgG4 isotype, and moreparticularly IgG1.

The present invention also provides Fabs, bi-specific antibodies andbi-specific antigen binding compounds, each produced according to themethods of the present invention, as well as host cells comprisingnucleic acids encoding the same. In particular, the present inventionprovides any of the Fabs, bispecific antibodies, nucleic acids or hostcells as exemplified in any of the Examples herein.

In another particular embodiment, the present invention provides abispecific antibody comprising a first IgG heavy chain, wherein saidfirst heavy chain comprises a variable domain comprising a lysinesubstituted at residue 39 (39K) and a glutamic acid substituted at theresidue which is four amino acids upstream of the lust residue of HFR3according to Kabat, a CH1 constant domain comprising an alaninesubstituted at residue 172 (172A) and a glycine substituted at residue174 (174G) and a CH3 constant domain comprising a lysine substituted atresidue 356 (356K) and a lysine substituted at residue 399 (399K); (b) afirst light chain, wherein said first light chain comprises a kappavariable domain comprising an arginine substituted at residue 1 (1R) andan aspartic acid substituted at residue 38 (38D), and a constant domaincomprising a tyrosine substituted at residue 135 (135Y) and a tryptophansubstituted at residue 176 (176W); (c) a second IgG heavy chain, whereinsaid second heavy chain comprises a variable domain comprising atyrosine substituted at residue 39 (39Y), a CH1 constant domaincomprising a WT sequence and a CH3 constant domain comprising anaspartic acid substituted at residue 392 (392D) and an aspartic acidsubstituted at residue 409 (409D); and (d) a second light chain, whereinsaid second light chain comprises a variable domain comprising anarginine substituted at residue 38 (38R) and a constant domaincomprising a WT sequence, wherein each of said first heavy chain andlight chain variable domains comprise three complementarity determiningregions (CDRs) which direct binding to a first antigen and furtherwherein each of said second heavy chain and light chain variable domainscomprise three complementarity determining regions (CDRs) which directbinding to a second antigen that differs from said first antigen.

BRIEF DESCRIPTION OF FIGURES

FIG. 1: Schematic diagrams of IgG (A), Fab-Fab (B) and IgG-Fab (C)design formats. The “*” in the IgG BsAb diagram indicates aheterodimerized antibody CH3 domain. The Fab-Fab format (B) could beprepared by expressing a polypeptide linker between the C-terminus ofthe HC of Fab 1 and the N-terminus of the HC or LC of Fab 2.Alternately, a polypeptide could connect the C-terminus of the LC of Fab1 and the N-terminus of the LC or HC of Fab 2. Similarly, the additionalFab in the IgG-Fab (C) could be linked to the N-terminus of the HC or LCor the C-terminus of the LC.

FIGS. 2 and 3: Surface Plasmon resonance (Biacore) traces demonstratingthe dual-binding behavior of the anti-HER-2/anti-EGFR (FIG. 2) andanti-cMET/anti-Axl (FIG. 3) IgG BsAbs: Bispecific binding of the FabRedesigned BsAbs (HE Designs (FIG. 2) and MA Designs (FIG. 3)) isevident by increases in signal during both injection cycles (300-540 secand 940-1180 sec). The monospecific MAbs pertuzumab IgG1 (pG1) andmatuzumab IgG1 (mG1) (FIG. 2) and METMAb IgG1 (METG1) and Anti-Axl IgG1(AxlG1) (FIG. 3) do not demonstrate this activity. The control moleculeswithout Fab redesigns (i.e., HE Control and MA Control), but harboringC_(H)3 heterodimerization mutations also demonstrate bispecific bindingactivity.

The general structure of an “antibody” is very well-known. For a fulllength antibody of the IgG type, there are four amino acid chains (two“heavy” chains and two “light” chains) that are cross-linked via intra-and inter-chain disulfide bonds. When expressed in certain biologicalsystems, e.g. mammalian cell lines, antibodies having unmodified humanFc sequences are glycosylated in the Fc region. Antibodies may beglycosylated at other positions as well. The subunit structures andthree-dimensional configurations of antibodies are well known. Eachheavy chain is comprised of an N-terminal heavy chain variable region(“V_(H)”) and a heavy chain constant region (“C_(H)”). The heavy chainconstant region is comprised of three domains (C_(H)1, C_(H)2, andC_(H)3) for IgG as well as a hinge region (“hinge”) between the C_(H1)and C_(H2) domains. Each light chain is comprised of a light chainvariable region (“V_(L)”) and a light chain constant region (“C_(L)”).The C_(L) and V_(L) regions may be of the kappa (“κ”) or lambda (“λ”)isotypes. IgG antibodies can be further divided into subclasses, e.g.,IgG1, IgG2, IgG3, IgG4, as appreciated by one of skill in the art.

The variable regions of each heavy chain-light chain pair associate toform binding sites. The heavy chain variable region (V_(H)) and thelight chain variable region (V_(L)) can be subdivided into regions ofhypervariability, termed complementarity determining regions (“CDRs”),interspersed with regions that are more conserved, termed frameworkregions (“FR”). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. CDRs of the heavy chain maybe referred to as “CDRH1, CDRH2, and CDRH3” and the 3 CDRs of the lightchain may be referred to as “CDRL1, CDRL2 and CDRL3.” The FRs of theheavy chain may be referred to as HFR1, HFR2, HFR3 and HFR4 whereas theFRs of the light chain may be referred to as LFR1, LFR2, LFR3 and LFR4.The CDRs contain most of the residues which form specific interactionswith the antigen

A wild type IgG antibody contains two identical fragments termed“fragment, antigen binding” (or Fab), each of which is composed of theV_(H) and C_(H)1 domains of one heavy chain and the V_(L) and C_(L)domains of a light chain. Each Fab directs binding of the antibody tothe same antigen. As used herein, the term “bi-specific antibody” or“IgG BsAb” refers to an IgG antibody comprising two distinct Fabs, eachof which direct binding to a separate antigen, and composed of twodistinct heavy chains and two distinct light chains. The V_(H) andC_(H)1 domains of one heavy chain associate with the V_(L) and C_(L)domains of one light chain to form a “first” Fab, whereas the V_(H) andC_(H)1 domains of the other heavy chain associate with the V_(L) andC_(L) domains of the other light chain to form a “second” Fab. Moreparticularly, the term “bi-specific antibody”, as used herein, refers toan IgG1, IgG2 or IgG4 class of bi-specific antibody. Even moreparticular, the term “bi-specific antibody” refers to an IgG1 or IgG4class of bi-specific antibody, and most particularly an IgG1 class.

The methods exemplified herein can be used to co-express two distinctFab moieties, for example Fabs with different Fv regions, with reducedmis-matching of respective HC/LC pairs. In addition to bi-specificantibodies and individual Fabs, the methods of the present invention canbe employed in the preparation of other bi-specific antigen bindingcompounds. As used herein, the term “bi-specific antigen bindingcompound” refers to Fab-Fab and IgG-Fab molecules. FIG. 1, includedherein, provides a schematic diagram of the structure of bi-specific,antibodies (IgG BsAb) as well as the Fab-Fab and IgG-Fab formatscontemplated by the methods and compounds of the present invention.

The methods and compounds of the present invention comprise designedamino acid modifications at particular residues within the variable andconstant domains of heavy chain and light chain polypeptides. As one ofordinary skill in the art will appreciate, various numbering conventionsmay be employed for designating particular amino acid residues withinIgG variable region sequences. Commonly used numbering conventionsinclude Kabat and EU index numbering (see, Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed, Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Other conventionsthat include corrections or alternate numbering systems for variabledomains include Chothia (Chothia C, Lesk A M (1987), J Mol Biol 196:901-917; Chothia, et al. (1989), Nature 342: 877-883), IMGT (Lefranc, etal. (2003), Dev Comp Immunol 27: 55-77), and AHo (Honegger A, PluckthunA (2001) J Mol Biol 309: 657-670). These references provide amino acidsequence numbering schemes for immunoglobulin variable regions thatdefine the location of variable region amino acid residues of antibodysequences. Unless otherwise expressly stated herein, all references toimmunoglobulin heavy, chain variable region (i.e., V_(H)) amino acidresidues (i.e. numbers) appearing in the Examples and Claims are basedon the Kabat numbering system, as are all references to V_(L) and C_(L)residues. All references to immunoglobulin heavy chain constant regionC_(H)1 and hinge appearing in the Examples and Claims are also based onthe Kabat system, whereas all references to immunoglobulin heavy chainconstant regions C_(H)2, and C_(H)3 are based on the EU Index numberingsystem. With knowledge of the residue number according to Kabat or EUIndex numbering, one of ordinary skill can apply the teachings, of theart to identify amino acid sequence modifications within the presentinvention, according to any commonly used numbering convention. Note,while the Examples and Claims of the present invention employ Kabat orEU Index to identify particular amino acid residues, it is understoodthat the SEQ IDs appearing in the Sequence Listing accompanying thepresent application, as generated by Patent In Version 3.5, providesequential numbering of amino acids within a given polypeptide and,thus, do not conform to the corresponding amino acid numbers as providedby Kabat or EU index.

However, as one of skill in the art will also appreciate, CDR sequencelength may vary between individual IgG molecules and, further, thenumbering of individual residues within a CDR may vary depending on thenumbering convention applied. Thus, to reduce ambiguity in thedesignation of amino acid residues within CDRs, the disclosure of thepresent invention first employs Kabat to identify the N-terminal (first)amino acid of the HFR3. The amino acid residue to be modified is thendesignated as being four (4) amino acid residues upstream (i.e. in theN-terminal direction) from the first amino acid in the reference HFR3.For example, Design A of the present invention comprises the replacementof a WT amino acid in HCDR2 with a glutamic acid (E). This replacementis made at the residue located four amino acids upstream of the firstamino acid of HFR3, according to Kabat. In the Kabat numbering system,amino acid residue X66 is the most N-terminal (first) amino acid residueof variable region heavy chain framework three (HFR3). One of ordinaryskill can employ such a strategy to identify the first amino acidresidue (most N-terminal) of heavy chain framework three (HFR3) from anyhuman IgG1 or IgG4 variable region. Once this landmark is determined,one can then locate the amino acid four residues upstream (N-terminal)to this location and replace that amino acid residue (using standardinsertion/deletion methods) with a glutamic acid (E) to achieve the“Design A” modification of the invention. Given any variable IgG1 orIgG4 immunoglobulin heavy chain amino acid query sequence of interest touse in the methods of the invention, one of ordinary skill in the art ofantibody engineering would be able to locate the N-terminal HFR3 residuein said query sequence and then count four amino acid residues upstreamtherefrom to arrive at the location in HCDR2 that should be modified toglutamic acid (E).

As use herein, the phrase “a/an [amino acid name] substituted at residue. . . ”, in reference to a heavy chain or light chain polypeptide,refers to substitution of the parental amino acid with the indicatedamino acid. For example, a heavy chain comprising “a lysine substitutedat residue 39” refers to a heavy chain wherein the parental amino acidsequence has been mutated to contain a lysine at residue number 39 inplace of the parental amino acid. Such mutations may also be representedby denoting a particular amino acid residue number, preceded by theparental amino acid and followed by the replacement amino acid. Forexample, “Q39K” refers to a replacement of a glutamine at residue 39with a lysine. Similarly, “39K” refers to replacement of a parentalamino acid with a lysine.

An antibody, Fab or other antigen binding compound of the presentinvention may be derived from a single copy or clone (e.g. a monoclonalantibody (mAb)), including any eukaryotic, prokaryotic, or phage clone.Preferably, a compound of the present invention exists in a homogeneousor substantially homogeneous population. In an embodiment, the antibody,Fab or other antigen binding compound, or a nucleic acid encoding thesame, is provided in “isolated” form. As used herein, the term“isolated” refers to a protein, peptide or nucleic acid which is free orsubstantially free from other macromolecular species found in a cellularenvironment.

A compound of the present invention can be produced using techniqueswell known in the art, e.g., recombinant technologies, phage displaytechnologies, synthetic technologies or combinations of suchtechnologies or other technologies readily known in the art. Inparticular, the methods and procedures of the Examples herein may bereadily employed. An antibody, Fab or antigen binding compound of thepresent invention may be further engineered to comprise frameworkregions derived from fully human frameworks. A variety of differenthuman framework sequences, may be used in carrying out embodiments ofthe present invention. Preferably, the framework regions of a compoundof the present invention are of human origin or are substantially human(at least 95%, 97% or 99% of human origin.) The sequences of frameworkregions of human origin may be obtained from The ImmunnglobulinFactsbook, by Marie-Paule Lefranc, Gerard Lefranc, Academic Press 2001,ISBN 012441351.

Expression vectors capable of directing expression of genes to whichthey are operably linked are well known in the art. Expression vectorscan encode a signal peptide that facilitates secretion of the desiredpolypeptide product(s) from a host cell. The signal peptide can be animmunoglobulin signal peptide or a heterologous signal peptide. Desiredpolypeptides, for example the components of the bi-specific antibodiesor Fabs prepared according to the methods of the present invention, maybe expressed independently using different promoters to which they areoperably linked in a single vector or, alternatively, the desiredproducts may be expressed independently using different promoters towhich they are operably linked in separate vectors. As used herein, a“host cell” refers to a cell that is stably or transiently transfected,transformed, transduced or infected with nucleotide sequences encoding adesired polypeptide product or products. Creation and isolation of hostcell lines producing a bi-specific antibody, Fab or other antigenbinding compound of the present invention can be accomplished usingstandard techniques known in the art.

Mammalian cells are preferred host cells for expression of the Fabs,bi-specific antibodies, or antigen binding compounds according to thepresent invention. Particular mammalian cells are HEK 293, NS0, DG-44,and CHO cells. Preferably, expressed polypeptides are secreted into themedium in which the host cells are cultured, from which the polypeptidescan be recovered isolated. Medium, into which an expressed polypeptidehas been secreted may be purified by conventional techniques. Forexample, the medium may be applied to and eluted from a Protein A or Gcolumn using conventional methods. Soluble aggregate and multimers maybe effectively removed by common techniques, including size exclusion,hydrophobic interaction, ion exchange, or hydroxyapatite chromatography.Recovered products may be immediately frozen, for example at −70° C., ormay be lyophilized.

The following Examples further illustrate the invention and providetypical procedures for carrying out various embodiments. However, it isunderstood that the Examples are set for the by way of illustration andnot limitation, and that various modification may be made by one ofordinary skill in the art. In addition, Lewis et al., Nature Biotech.,32(2); February 2014, provides additional illustration of embodiments ofthe present invention.

Example 1. Computational Design to Identify Modifications ofC_(H)1/C_(L) Interface Residues that Discriminate Between Designed andNative Immunoglobulin C_(H)1/C_(L) Interfaces

Residues for initial modification at the C_(H)1/C_(L) interface areselected using a combination of computational and rational designstrategies. Using a crystal structure of the IgG1/λ Fab (PDB ID 3TV3(see, Lewis, S. M. and Kuhlman, B. A. (2011), PLoS ONE 6(6): e20872)),trimmed to heavy chain residues 112-228 and light chain residues106A-211, the Rosetta software suite and related modeling applicationsare employed to evolve potential sequences for modification according toa desired fitness function (see, Kaufmann et al. (2010), Biochemistry49; 2987-2998; Leaver-Fay et al. (2011), Methods Enzymol. 487; 545-574;Kuhlman et al. (2003), Science 302(5649); 1364-1368; and Leaver-Fay etal. (2011), PLos ONE 6(7): e20937). Briefly, Rosetta calculates afitness score and binding energy based on a weighted sum of energypotentials treating phenomena such as van der Waals forces and hydrogenbonding forces. Overall, the summations of these different parametersare measured in units known as the Rosetta Energy Unit (REU). Thesevalues are interpreted as free energies, but are not directlytranslatable into typical units of energy.

Using a fitness function which favors the binding and stability ofdesigned-C_(H)1/designed-C_(L) complexes and disfavors binding affinityof undesired designed-C_(H)1/WT-C_(L) and WT-C_(H)1/designed-C_(L)complexes; initial sequence modifications resulting in bindingorthogonality for designed C_(H)1/C_(L) pairs are identified.

The identified mutations are subjected to computational re-docking ofthe C_(H)1/C_(L) complex using RosettaDock via RosettaScripts (see,Chaudhury et al. (2011). PLoS ONE 6(8): e2247 and Fleishman et al.(2011), PLoS ONE 6(6): e20161). This allows determination of optimalbinding positions for designed complexes and allows comparison withbinding energies of the similarly docked WT complexes and the undesireddesigned-C_(H)1/WT-C_(L) and WT-C_(H)1/designed-C_(L) complexes. Adeficit in the computational total score and binding energies for theseundesired complexes predicts they will bind weakly to one anothercompared to the WT-C_(H)1/WT-C_(L) and designed-C_(H)1/designed-C_(L)complexes. Total energies are calculated using a Rosetta standardscorefunction, “Score12 prime.” (see, Leaver-Fay et al. (2013), MethodsEnzymol. 523; 109-143). Binding energies are calculated as the change infree energy (ΔG) separated score as reported by Rosetta'sInterfaceAnalyzer tool (see, Lewis, S. M. and Kuhlman, B. A. (2011),PLoS One 6(6): e20872). Representative design constructs and theircorresponding total score and binding energies are provided in Table 1.

TABLE 1 Rosetta multi-state computational design results. Con- HC mut/LCmut HC mut/LC wt HC wt/LC mut struct total binding total binding totalbinding (C_(H)1/C_(λ)) Mutations^(b) score^(a) energy score energy scoreenergy WT None −359 −29 — — — — 1.0 H_F174T −357 −26 −353 −23 −355 −27H_V190F L_L135F 2.1 H_F174G −355 −26 −352 −23 −347 −22 L_L135A L_S176W5.0 H_D146K −359 −29 −355 −28 −359 −29 L_K129D 1.0 + H_D146K −357 −28−352 −25 −353 −24 5.0 H_F174T H_V190F L_L135F L_K129D ^(a)Units for thefitness score are called Rosetta Energy Units (REUs). ^(b)Mutations aredesignated by first identifying the heavy chain (H) or light chain (L),followed by the one letter abbreviation for the parental amino acid, theamino acid residue number and the one letter abbreviation for thereplacement amino acid (For example, H_F174T indicates that residue 174of the heavy chain is modified from a phenylalanine (F) to a threonine(T).

More than forty discrete initial designs, falling into about twentydifferent design paradigms (i.e., mutations with different amino acidsubstitution combinations and different residue positions), wereidentified after filtering of many more computationally-generatedsequences. Select design paradigms were synthesized and furtherinterrogated experimentally either in a full-length IgG1/λ construct oran IgG1/λ construct that lacks variable domains, each as described below(see Tables 2 and 3). Based on those experiments, three designs, Design1.0, Design 2.1 and Design 5.0 and Design 1.0+5.0 demonstrated goodbiophysical properties and thermodynamic discrimination fordesigned-C_(H)1/designed-C_(L) association over designed-C_(H)1/WT-C_(L)or WT-C_(H)1/designed-C_(L) association as demonstrated in thermalchallenge assays (described below).

Synthesis of Test Articles.

To test the designs, a pertuzumab (see, Nahta et al. (2004), Cancer Res.64; 2343-2346) human IgG1 with a human chimeric kappa V_(L) and lambdaC_(L) (C_(λ)) is created. Briefly, the pertuzumab V_(H) domain insert isgenerated using a PCR-based overlapping oligonucleotide synthesisprocedure (Casimiro et al. (1997), Structure 5; 1407-1412) using thesequence from the published crystal structure (Franklin et al. (2004),Cancer Cell 5; 317-328). The insert contains appropriate AgeI and NheIrestriction sites that enable it to be ligated directly into alinearized in-house pE vector (Lonza) containing an IgG1 constant domainsequence. The pertuzumab V_(L) gene is also generated using overlappingoligonucleotide synthesis. A DNA sequence encoding the V_(L) domainfused to C_(λ) is constructed using PCR and an in-house plasmid templatecontaining the C_(λ) sequence. Both 5′ and 3′ flanking oligonucleotidesand two internal primers are designed to anneal the C-terminus of thepertuzumab V_(L) domain to the N-terminus of C_(λ). The light chaininsert is designed with HindIII and EcoRI restriction sites for directligation into a linearized in-house pE vector (Lonza) with a selectableGS marker system. Each pE mammalian expression vector is engineered tocontain a common mouse antibody light chain signal sequence that istranslated in-frame as part of the expressed protein and cleaved priorto secretion. All ligation constructs are transformed into E. colistrain TOP 10 competent cells (Life Technologies). Transformed bacterialcolonies are picked, cultured, and the plasmids are prepped. Correctsequences are confirmed by DNA sequencing. The encoded sequences of themature heavy chain and light chain proteins are given by SEQ ID NO:1 andSEQ ID NO:2, respectively.

To further interrogate the ability of C_(H)1/C_(L) designs to provide anew and specific interface that discriminates from WT C_(H)1/C_(L)interfaces, it is useful to remove the variable domains, which, ifpresent, add complexity to the data interpretation. Therefore, humanIgG1 and human, lambda constructs lacking variable genes (V_(H) andV_(L)) are constructed. A recombinase-based subcloning strategy is usedto remove the variable genes. Briefly, for the heavy chain plasmid, twodouble stranded oligonucleotides that encompass 15 base pairs 5′ of anXhoI site through the common mouse antibody light chain signal sequencefollowed immediately by a NheI site and 15 flanking base pairs (encodingthe N-terminus of the IgG1 C_(H)1 domain for efficient recombination)are chemically synthesized. This double stranded oligonucleotide pairhas the V_(H) domain deleted. For the light chain plasmid, two doublestranded oligonucleotides that encompass 15 base pairs 5′ of a BamHIsite through the common mouse antibody light chain signal sequencefollowed immediately by an XmaI and 15 flanking base pairs (encoding theN-terminus of the lambda C_(L) domain for efficient recombination) arechemically synthesized. This double stranded oligonucleotide pair hasthe V_(L) domain deleted. The variable genes are digested out of theparental pertuzumab heavy chain and light chain plasmids using theXhoI/NheI and BamHI/XmaI enzymes, respectively. The correspondingoligonucleotide pairs are inserted into the linearized plasmids usingthe recombinase-based In-Fusion HD Cloning kit (Clontech) according tothe manufacturer's protocol. The sequences of the heavy chain and lightchain IgG1/λ constructs lacking variable domains are given by SEQ ID NO:3 and SEQ ID NO: 4, respectively.

For generating small sets of mutations, the QuikChange II Site DirectedMutagenesis Kit (Agilent) may be used following the instructionsprovided by the manufacturer. For generating large sets of mutations(typically >3 mutations per chain), a gene synthesis strategy may beemployed (G-blocks, IDT). The synthesized genes are designed to becompatible with the heavy chain and light chain construct lackingvariable domains (described above). However, within the heavy chainconstruct, an Xho I site upstream of the common mouse light chain signalsequence is deleted using site directed mutagenesis and a new Xho I siteis generated at the C-terminus (3′ end) of the coding region of theC_(H)1 domain. Synthesized genes are ligated to the heavy chain plasmidusing the NheI and new XhoI restriction sites. Synthesized genes areadded to the light chain plasmid using the BamHI and EcoRI restrictionsites.

Protein Expression and Characterization.

Each plasmid is scaled-up by transformation into TOP10 E. coli, mixedwith 100 mL luria broth in a 250 mL baffled flask, and shaken O/N at 220rpm. Large scale plasmid purifications are performed using the BenchPro2100 (Life Technologies) according to the manufacturer's instructions.

For protein production, plasmids harboring the heavy chain and lightchain DNA sequences are transfected (1:2 plasmid ratio for the heavychain and light chain plasmids, respectively) into ITEK293F cells usingFreestyle transfection reagents and protocols provided by themanufacturer (Life Technologies). Transfected cells are grown at 37° C.in a 5% CO₂ incubator while shaking at 125 rpm for 5 days. Secretedprotein is harvested by centrifugation at 10 K rpm for 5 min.Supernatants are passed through 2 μm filters (both large scale and smallscale) for purification. Small scale (1 mL) purifications are performedby directly incubating 1 mL transfected supernatant with 100 μLresuspended, phosphate buffered saline (PBS) washed-Protein G magneticbeads (Millipore). Beads are washed 2-times with PBS and 1-time with10-fold diluted PBS. Protein is eluted from the beads by adding 130 μL0.01 M Acetate, pH 3.0. After harvesting, the eluants are immediatelyneutralized by adding 20 μL 0.1 M Tris, pH 9.0. The concentration of thepurified proteins are determined by measuring the absorbance of thesolutions at 280 nm using a NanoDrop UV-Vis spectrophotometer fromThermoScientific (Grimsley, G. R. and Pace, C. N. (2004), Curr. Protoc.Protein Sci., Ch. 3 (3.1)).

Identities of representative transfected plasmids and the sequences theygenerate are provided in Table 2. Methods for characterization and theresulting data for each of these heavy chain/light chain designs aredescribed below.

TABLE 2 Sequence composition of the C_(H)1/C_(L) interface specificitydesigns based on Rosetta. Heavy Light Chain Chain SEQ ID SEQ ID DesignMutations NO: NO: WT pertuzumab None 1 2 IgG1/λ WT IgG1/C_(λ) None 3 4lacking V-genes Design 1.0^(a) HC_F174T, HC_V190F, 5 6 LC_L135F Design2.1^(b) HC_F174G, LC_L135A, 7 8 LC_S176W Design 5.0^(a) HC_D146K,LC_K129D 9 10 Design 1.0 + 5.0^(b) HC_D146K, HC_F174T, 11 12 HC_V190F,LC_K129D, LC_L135F ^(a)In the pertuzumab IgG1/λ format. ^(b)In theIgG1/λ format lacking variable domains

The proteins may be characterized using multiple methods, as describedherein. For example, sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) analyses are used to evaluate expression andassembly of the HC and LC components. SDS-PAGE may be performed usingNuPAGE® Novex 4-12% BisTris gels according to manufacturer protocols(Life Technologies). Approximately 15 μL of purified material from themagnetic bead purifications (see above) are loaded in each well. Forreduced samples, 10% 0.5 M DTT in H₂O is added. Protein bands on thegels are detected using a SimplyBlue™ Safe Stain (Life Technologies).

Enzyme-linked immunosorbent assays (ELISAs) for the detection ofthermochallenged protein samples may also be performed to compare thestability of the designed samples against the wild-type control,proteins and the mis-matched designs. T₅₀ values, defined as thetemperature at which 50% of ELISA signal that detects protein activityremains, after heating at elevated temperature for a specified period oftime, can be determined for each sample to compare the stability of thedesigned HC/LC constructs relative to the stability of wild-typecontrols and mis-matched pairs. For the pertuzumab IgG1/λ proteins,96-well U-bottom high protein binding 96-well plates (Greiner bio-one,cat #650061) are coated overnight at 4° C. with 100 μL/well of apolyclonal anti-human C_(λ) antibody (Southern Biotech, cat #2070-01) at2 μg/mL in a 0.05 M NaHCO₃ buffer, pH 8.3. The plates are then washedfour times with PBS with 0.02% Tween80 (PBST) and blocked for 1 hr withcasein (Thermo, cat #37528) at 37° C. The plates are washed againfollowed by the addition of isolated HEK293F culture supernatantscontaining the pertuzumab IgG1/λ protein designs (100 μL/well). Aliquotsof each supernatant are pre-exposed to various temperatures for 1 hrusing a PCR instrument with a 25° C. thermal gradient window. Thethermal challenged pertuzumab IgG1/λ proteins (with or without mutationsin the C_(H)1/C_(L) domains) are incubated on the plate for 1 hr at 37°C. The plates are then washed and 200 ng/mL in-house biotinylated humanHER-2-Fc (R&D systems, cat #1129-ER-020) is added at 100 μL/well(diluted in casein) for 1 hr at 37° C. The plates are washed againfollowed by the addition of streptavidin-HRP (Jackson Immunoresearch,cat #016-030-084) diluted 1:2000 in casein. The plates are then washedand SureBlue Reserve TMB 1-component substrate (KPL, cat #53-00-01) isadded at 100 μL/well. The reaction is allowed to proceed for 5-15minutes then quenched by the addition of 1% H₃PO₄. The absorbance at 450nm is read using a SpectraMax 190 UV plate reader (Molecular Devices). Asimilar procedure is followed for the detection of thermochallengedIgG1/λ minus variable gene proteins using a polyclonal anti-human C_(H)1antibody (2 μg/mL in casein; Meridian Life Sciences, cat #W90075C) tocapture proteins from the supernatants and a HRP-labeled polyclonalanti-human C_(λ) antibody (1:2000 dilution in casein; Southern Biotech,cat #2070-05) for detection (replacing the HER-2-Fc-biotin andstreptavidin-HRP).

Characterization results for each of the designs depicted in Table 2 areprovided in Table 3 below. Three designs, Design 2.1, Design 5.0, andDesign 1.0+5.0 were found where the thermal stability of the designedHC/designed LC C_(H)1/C_(L)-containing IgG protein was superior bothcomputationally and experimentally over at least one of the mismatchedDesigned HC/WT LC or WT HC/Designed LC pairs (Table 3). In some cases(including Designs 2.1 and 1.0+5.0), the designs were tested in theIgG1/C_(λ) protein lacking variable domains. For both Design 1.0+5.0 andDesign 2.1, a clear preference for the Designed HC/Designed LC paircould be seen by SDS-PAGE analysis as evident by a strong band at ˜100kDa resembling that of the wild-type HC/LC pairs. The Design 1.0+5.0mismatched pairs (i.e., Designed HC/WT LC and WT HC/Designed LC)expressed too poorly to be seen on an SDS-PAGE gel. The Design 2.1mismatched pairs demonstrated additional banding below the main bandindicative of unassembled protein.

TABLE 3 Characterization of designed C_(H)1/C_(L) antibody proteins.Titer^(a) Titer^(a) Titer^(a) ((HCmut/ ((HCwt/ T₅₀ T₅₀ (ConstructLCwt^(b)) LCmut^(b)) SDS- (° C.) (° C.) vs. WT vs WT vs WT PAGE T₅HCmut/ HCwt/ Construct Mutation control) Control) Conrrol) Assembly^(c)(° C.) LCwt LCmut WT None 1.0 n.d.^(d) n.d. + 66 n.d. n.d. PertuzumabIgG1/λ WT None 1.0 n.d. n.d. + 78 n.d. n.d. IgG1/C_(λ) minus V-genesDesign 1.0^(e) HC_F174T 1.0 3.1 − + 67 67 n.d. HC_V190F LC_L135F Design2.1^(f) HC_F174G 1.1 0.2 0.5 + >75 69 74-75 LC_S176W LC_L135A Design5.0^(e) HC_D146K 0.5 0.75 0.5 + 67 60 67 LC_K129D Design 1.0 + HC_D146K0.6 0.0 −0.02 + 73 <45 53 5.0^(f) HC_F174T HC_V190F LC_K129D LC_L135F^(a)Titers are determined based on the protein G magnetic bead purifiedprotein recoveries as described above. Titer values are given as a ratioof expression of the indicated designed contruct or mis-matched pair(i.e. HCmut/LCwt or HCwt/LCmut) relative to expression of theappropriate WT control. ^(b)The mis-matched pairs were expressedseparately by co-transfection of a designed HC or LC with a wild-type LCor HC, repectively. ^(c)Assembly denoted by single band at 100 kDa bySDS-PAGE (+). Partial assembly denoted by multiple bands on SDS-PAGEincluding 100 kDa band (+/−). No band at 100 kDa on SDS-PAGE denoted by(−). ^(d)n.d. = not determined. ^(e)Determined in the pertuzumab IgG1format. ^(f)Determined in the IgG1/λ minus variable genes format.

The characterization results demonstrate that Design 1.0+5.0 maintains ahigh affinity and stable C_(H)1/C_(L) interface with low capability forrecognizing native immunoglobulin C_(H)1/C_(L) domains. Additionally,Design 2.1 demonstrates high stability and specificity for itself whilediscriminating against binding to native (wt) immunoglobulinC_(H)1/C_(L) domains.

Analytical size exclusion chromatography with in-line light staticscattering (SEC/LS) is another characterization tool, used to confirmthat the heterotetrameric HC/LC antibody complexes associate properlyand continue to demonstrate monodisperse biophysical behavior. SEC/LSmay be performed for each sample using 30-80 μL of purified eluant fromthe small scale-purification described above (concentrations ˜0.1-0.4mg/mL). For SEC/LS, the proteins are injected onto a Sepax Zenix SEC 200analytical HPLC (7.8×300 mm) column equilibrated in 10 mM phosphate, 150mM NaCl, 0.02% NaN₃; pH 6.8, using an Agilent 1100 HPLC system. Staticlight scattering data for material eluted from the SEC column arecollected using a miniDAWN TREOS static light scattering detectorcoupled to an Optilab T-rEX in-line refractive index meter (WyattTechnologies). UV data are analyzed using HPCHEM (Agilent). Proteinmolecular weights are determined by their static light scatteringprofiles using ASTRA V (Wyatt Technologies). SEC/LS analysis indicatesthat all of the proteins described in Table 3 were shown to be highlymonodisperse with soluble aggregates <5% and indistinguishable from thewild-type IgG and IgG minus variable domain constructs.

Example 2. Optimization of Design 2.1

Design 2.1, as described in Example 1, is further optimized to improvespecificity of assembly of the designed C_(H)1/C_(L) interface versus aWT C_(H)1/C_(L) interface. To aid in the design process, in-housecrystal structures of Design 1.0+5.0 and Design 2.1 are first solved.

To generate protein for crystallography, isolated C_(H)1/C_(λ) proteins(disulfide linked) are produced in E. Coli. The isolated C_(H)1/C_(λ)proteins (no variable-genes or IgG-Fc) are subcloned into the pET-DUETplasmid from Novagen. The C_(H)1 insert (with a pelB signal sequence forsecretion into the oxidative periplasmic environment) is synthesized(with a hexahistidine C-terminal tag) using overlapping PCR andsubcloned into cassette 1 of the plasmid using the NheI and BamHI sites.The C_(λ) insert is similarly synthesized and inserted between the NdeIand XhoI sites (no his tag). Designs 1.0+5.0 and Design 2.1 aregenerated from the WT plasmid using QuikChange II mutagenesis (Agilent).Each plasmid is transformed into CodonPlus BL21(DE3) chemicallycompetent cells (Agilent) for expression. For each protein preparation,transformed and pre-cultured cells are used to inoculate 2×1.4 L luriabroth supplemented with 100 μg/mL carbenicillin and 35 μg/mLchloramphenicol. The cultures are allowed to shake at 220 rpm at 37° C.until the OD₆₀₀ reached 1-1.5. At this stage, the culture temperature isreduced to 30° C. and 1 mM Isopropyl-1-thio-β-D-galactopyranoside isadded. The cultures are allowed to grow for 3-4 hrs and harvested bycentrifugation for 20 min at 4000 g. The proteins are resuspended in 50mL of a periplasmic extraction buffer (500 mM sucrose, 100 mM Tris, pH8, 1 mM EDTA, and 100 μg/mL hen-egg white lysozyme). The extractedproteins are diluted 10-fold into a 10 mM citrate, 10 mM NaCl buffer, pH5.5 and passed over a 5 mL SP Sepharose FF HiTrap cation exchange column(GE Healthcare) at 5 mL/min using an AKTA Explorer (GE Healthecare). Theproteins are eluted from the column using a gradient up to 0.7 M NaCl.The proteins are dialyzed into PBS and captured onto a Ni-SepharoseHiTRAP affinity column. The proteins are then eluted using a gradient upto 0.3 M imidazole. The proteins are then concentrated to ˜3-10 mg/mLusing VivaSpin6 centrifugal devices, dialyzed into 10 mM Tris, 100 mMNaCl, pH 8.0 and filtered.

For crystallography, the purified proteins are screened using the vapordiffusion crystallization method, whereby protein is first mixed withwell solution and deposited in a small chamber with well solution,sealed and allowed to equilibrate with well solution, concentrating bothprotein and precipitating reagents in the protein drop. Such screens areconducted in a 96-well format (Intelli-plates, Art Robins Instrument)using the commercially available screens: PEGs, PEGs II, ComPAS,Classics, Classics II Suites (Qiagen). The initial setup utilizes aPhoenix robot (Art Robins Instrument), which deposits 0.3 μL of proteinon 0.3 μL of well solution.

For the WT C_(H)1/C_(λ) protein, initial screening is performed at 6mg/ml protein. Protein crystals grew after 2 days in the followingcondition: 15% Ethanol/50% MPD/10 mM Sodium Acetate. These initialcrystallization conditions are optimized in a set of vapor diffusionexperiments where the concentration of the two components is varied,while the third is kept constant. The optimization experiments areconducted in 48-well format Intelli-plates (Art Robins Instrument). Theoptimization experiments result in thin needle-shaped crystalsdiffracting below 3 Å resolution.

Next, protein is re-screened at higher concentration of 9.2 mg/ml.Streak seeding using the needle-shaped crystals from the previous stepis performed on, the following day. Streak seeding is a method promotingcrystal nucleation by providing a ready-made nucleus (seed crystals) toassist nucleation and facilitate the growth of ordered crystals. Thistechnique results in a new crystallization condition: 30% PEG4K.Optimizing of this condition is performed at 21° C. by varying the PEG4Kconcentration, the size of the drop containing the protein and reservoirsolution mixture, and the ratio of the protein and reservoir content inthe drop. Crystallization drops are seeded and crystals appeared on thenext day, growing to the final size within 3 days. Crystals aretransferred to a cryo-protection solution of the reservoir solution withPEG4K increased by 10% and supplemented by 20% PEG400 and flash frozenby immersion in liquid nitrogen before shipping to the Advanced PhotonSource for data collection.

Design 1.0+5.0 is similarly screened and streak-seeded using the WTcrystal seeds. Intergrown needle-shaped crystals appeared after 3 daysat two conditions: 20% Isopropanol/20% PEG4K/100 mM tri-Sodium Citrateand 33% PEG6K/10 mM tri-Sodium Citrate. Both conditions are optimized,using the technique described above, resulting in single crystals forthe optimization of the 2^(nd) condition. Final Design 1.0+5.0 singlecrystals are grown at 21° C. by mixing 1.2 μL of 3.9 mg/ml protein and1.2 μL of reservoir solution, containing 40% PEG6K and 10 mM tri-SodiumCitrate dehydrate. Crystals are cryo-protected using reservoir solutionincreased by 10% PEG6K content and supplemented by 20% of EthyleneGlycol.

Design 2.1 crystallizes directly in conditions developed for Design1.0+5.0. Single crystals are grown at 21° C. by mixing 1.5 μL of 5.1mg/ml protein with 1.5 μL of reservoir, containing 39% PEG6K and 10 mMtri-Sodium Citrate. Crystals are cryoprotected using the same techniqueas for Design 1.0+5.0.

The conformations of the designed residues observed in the computationalmodel structure and the experimental crystal structure of Design 1.0+5.0were very similar (i.e., the designed residues adopted the sameconformations in the model and the experimental in-house crystal ofDesign 1.0+5.0). However, the designed residues of Design 2.1 showed asubstantially different orientation in the computational model than inan in-house crystal structure. Specifically, the light chain designedresidue 176W and heavy chain native residue H172 of the in-house crystalstructure did not adopt the conformations of the Rosetta computationalmodel. The crystal also revealed a trans-cis isomerization between heavychain residues G174 (mutated from F) and P175. This structuralrearrangement was unanticipated by the model, but also not within themodeling degrees of freedom. Further modifications for Design 2.1 weregenerated following computational design methods essentially asdescribed in Example 1, but using the in-house crystal structure ofDesign 2.1 as the starting point in place of the original 3TV3 crystalstructure model.

Following molecular biology procedures essentially as described inExample 1, modifications to Design 2.1 are constructed in the IgG/Cλconstruct lacking variable domains. Table 4 provides identities andcorresponding mutations for representative further-optimized designs ofDesign 2.1.

TABLE 4 Optimization of Design 2.1. HC SEQ LC SEQ Design C_(H)1/C_(L)Mutations ID NO: ID NO: Design HC_H172A, HC_F174G, LC_L135F, 18 152.1.3.2 LC_S176W Design HC_H172S, HC_F174G, LC_L135Y, 19 20 2.1.3.3LC_S176W Design HC_H172A, HC_F174G, LC_L135Y, 18 20 2.1.3.3a LC_S176W

The optimized Design 2.1 proteins are expressed using the transientHEK293F system as described in Example 1. Each of the designs are testedfor thermal stability by thermal challenge at temperatures ranging from70-95° C. using the methodology as described in Example 1. The sustainedpresence of each design protein after heating is determined using theIgG1/λ minus variable gene ELISA also as described in Example 1. Resultsof the thermal challenge stability test for representative optimizeddesigns of Design 2.1 as well as WT IgG1/C_(λ) (no V_(H)/V_(L)) andDesign 2.1 are provided below in Table 5.

TABLE 5 Optimization of Design 2.1 Design C_(H)1/C_(L) Mutations T₅₀ (°C.) WT IgG1/C_(λ) None 78.1 ± 0.5  (no V_(H)/V_(L)) Design 2.1 HC_F174G,LC_S176W, LC_L135A >75 Design 2.1.3.2 HC_H172A, HC_F174G, LC_L135F, 81.4± 0.5  LC_S176W Design 2.1.3.3 HC_H172S, HC_F174G, LC_L135Y, 77.7 ± 0.5 LC_S176W Design 2.1.3.3a HC_H172A, HC_F174G, LC_L135Y, 83 ± 2  LC_S176W

The thermochallenge data in Table 5 indicates Design 2.1.3.2, Design2.1.3.3 and Design 2.1.3.3a were the most stable constructs.

To further assess specificity of optimized designs of Design 2.1, a massspectrometry (MS) method to directly measure the specificity versus theWT C_(H)1/C_(L) domains is performed. Briefly, at the 2 mL transfectionscale, a designed C_(L) domain is co-transfected with a WT C_(L) domainand either a designed heavy chain or a WT heavy chain. In this way thedesigned and WT C_(L) proteins directly compete with one another duringprotein expression for binding to the heavy chain protein and secretioninto the cell media. The assembled IgG/C_(L) minus variable genesproteins are purified automatically using an Agilent 1100 series HPLCwith autosampler and sample collector. The proteins are captured on a PG(protein G) HPLC column (Applied Biosystems, Cat #2-1002-00) at 2mL/min, washed with phosphate buffered saline (PBS) and eluted withdistilled deionized H₂O, 0.2% formic acid. The protein eluants areconcentrated/dried on a Labconco CentriVap Speedvac for 3 hrs at 40° C.under vacuum. The proteins are resuspended in 100 μL distilled deionizedwater and neutralized with 20 mL 0.1 M Tris pH 8.0 (MediaTech, Inc., Cat#46-031-CM). The intermolecular disulfides (HC/LC and HC/HC) are reducedby the addition of 10 μL freshly solubilized 1 M dithiothreitol (DTT,Sigma, Cat #43815-1G). The samples are sequentially collected using anAgilent 1100 series HPLC with an autosampler and captured onto a reversephase C4 analytical column for desalting using a water, 0.2% formic acidmobile phase. After separation of the protein from its buffercomponents, the proteins are bumped from the C4 column using a 10%/90%water/acetonitrile pulse (both with 0.2% formic acid) and injected intoan Agilent 6210 time-of-flight liquid chromatography/masse spectrometrysystem molecular weight analyser. Theoretical mass-averaged molecularweights of the light chain and heavy chain components are determinedusing the GPMaw program (v. 8.20). The two separate light chains of thecompetition experiment can be easily discriminated from one anotherbased on their different molecular weights. The relative counts of theionized light chains hitting the detector are used to quantify the ratioof designed C_(L) and WT C_(L) protein that is bound to the heavy chaincomponent and this data is shown in Table 6.

TABLE 6 Results of the competition LC specificity LC/MS assay. Total % %Expres- Assembly^(a) Assembly^(a) sion LC1 LC2 HC (LC1/HC) (LC2/HC)(μg/mL) 2.1.3.2 Cλ WT Cλ WT HC 11 89 73 2.1.3.2 Cλ WT Cλ 2.1.3.2 HC 7822 76 2.1.3.3a Cλ WT Cλ WT HC 10 90 50 2.1.3.3a Cλ WT Cλ 2.1.3.3a HC 99 1 80 2.1.3.3a Cλ WT Cκ WT HC 8 ± 3  92 ± 3  63 ± 20 2.1.3.3a Cλ WT Cκ2.1.3.3a HC 100 ± 0.2  0.2 ± 0.2 75 ± 20 ^(a)“% Assembly” is calculatedas described in the legend for Table 11, below

Results of the LC/MS competition experiments indicate significantspecificity versus WT sequences is obtained using Designs 2.1.3.2 and2.1.3.3a. Both Design 2.1.3.2C_(λ) and Design 2.1.3.3aC_(λ) proteinssignificantly out-competed WT C_(λ) (and WT C_(κ)) for binding to theirheavy chain protein counterpart containing the 2.1.3.2 or 2.1.3.3a heavychain mutations. Conversely, Design 2.1.3.2 and Design 2.1.3.3a C_(λ)proteins did not compete well with WT Cλ (and WT C_(κ)) for binding tothe WT HC protein. Design 2.1.3.3a C_(λ) provided slightly betterspecificity than Design 2.1.3.2C_(λ) in the experiment.

Example 3. Designs to Generate Designed V_(H) and V_(L) Domains thatBind One Another Stronger than They Bind WT V_(L) or V_(H) Domains,Respectively

To further optimize specific heavy chain-light chain assembly whenco-expressing two Fabs, variable heavy chain/variable light chain(V_(H)/V_(L)) modifications are designed. V_(L) position 1 and V_(H)position 62 are first identified for modification. V_(L) position 1 ismodified to the positively charged residue arginine (R) and V_(H)position 62 is modified to the negatively charged residue glutamic acid(E). This combination of charge modifications is denoted Design A. Asecond design modified V_(L) position 38 and V_(H) position 39 togenerate a charge pair (V_(L_)Q38D and V_(H_)Q39K) at the site of theformer hydrogen bonding interaction. This charge pair introduction isdenoted Design B.

Design A, Design B, and a combination of Design A and B, denoted DesignAB, are introduced into the plasmids for the pertuzumab light chain andheavy chain plasmids for mammalian expression. Plasmids containingDesign 1.0+5.0 (with WT petuzumab V_(H) and V_(L)) modifications arealso constructed to generate heavy chains and light chains topotentially improve specificity of the variable domain designs overpairing with fully wild type heavy and light chains. The mutations areintroduced using the using the QuikChange II mutagenesis kit (Agilent)according to the manufacturer's protocols constructs. The methodsfollowed for plasmid production and purification are essentially asdescribed in Example 1. The identities and corresponding sequences ofDesign A, Design B, and Design AB are provided in Table 7.

TABLE 7 SEQ ID NOs of Variable Domain and Constant Domain Designs in thepertuzumab HC and LC. Full-length HC/LC IgG HC SEQ LC SEQ DesignMutations ID NO: ID NO: Variable Domain Designs^(a) Design A HC_R62E,LC_D1R 21 22 Design B HC_Q39K, LC_Q38D 23 24 Design AB HC_R62E, HC_Q39K,25 26 LC_D1R, LC_Q38D Constant Domain Designs^(b) Design HC_D146K,HC_F174T, 27 28 1.0 + 5.0 HC_V190F, LC_K129D, LC_L135F ^(a)The VariableDomain Designs contain the indicated mutations in the V_(H) and V_(L)domains and WT pertuzumab sequences in the C_(H) and C_(L) domains.^(b)The Constant Domain Designs contain the indicated mutations in theC_(H) and C_(L) domains and WT pertuzumab sequences in the V_(H) andV_(L) domains.

Each of the designed heavy chain and light chain plasmids areco-expressed transiently in HEK293F essentially as described inExample 1. To probe for specificity, the designed heavy chains and lightchains are also expressed with a mismatched light chain or heavy chainas a control to probe for specificity. The mismatched heavy chain andlight chain contain WT V_(H) and V_(L) domains and the Design 1.0+5.0 inthe constant domains to add additional specificity (denoted in Table 8below as HC(WT15) and LC(WT15), respectively). The expressionsupernatants are subjected to a thermal challenge by incubation for 1 hrat temperatures ranging from 45-75° C. and tested for binding hHER-2-Fcusing an ELISA (methods described in Example 1). This ELISA format issensitive to the stability of the variable domains. Additionally, allproteins are purified from their supernatants using the protein Gmagnetic bead protocol, essentially as described in Example 1, andformed fully assembled IgG molecules. In each of the cases (Design A andDesign B), the apparent thermal stability of the matched heavy chain andlight chain pairs is significantly higher than the mismatched pairs. Theresult indicate that the matched designed pairs are thermodynamicallyfavored over the mismatched pairs and provides a thermodynamic basis forthe specific, association of the Design A heavy chain and light chainfor themselves and the Design B heavy chain and light chain forthemselves over the association with the heavy chain and light chainscontaining WT variable domains. Combining the designs into a singleconstruct, Design AB, further improved the thermodynamic specificity andresulted in reduced expression of the mismatched pairs. Results for thethermo-challenge testing are provided in Table 8.

TABLE 8 Summary of the thermochallenge data with Design A, Design B, andDesign AB. Full Length HC/LC HC/LC SEQ IgG Construct ID NO: T₅₀ (° C.)Matched pair Constructs HC_Design A/LC_Design A 21/22 63.6 ± 0.6 HC_Design B/LC_Design B 23/24 59.0 ± 1.0  HC_Design AB/LC Design AB25/26 59.3 ± 0.5  Mis-matched pair Constructs HC (WT15)/LC Design A27/22 52.8 ± 0.6  HC (WT15)/LC Design B 27/24 46.7 ± 0.2  HC (WT15)/LCDesign AB 27/26 <45 HC_Design A/LC (WT15) 21/28 58.1 ± 0.2  HC_DesignB/LC (WT15) 23/28 53.6 ± 0.4  HC_Design AB/LC (WT15) 25/28 57.9 ± 0.3 

The results in Table 8 indicate that making any of the threecompensatory charge pair modifications described above maintains thethermostability of heavy chain/light chain assembly within the designconstructs while reducing the thermostability of the mis-matcheddesigns. These variable domain designs can be added to the constantdomain designs described in Examples 1 and 2 above to help improvespecific heavy chain/light chain assembly.

Example 4. Multi-State Computational Designs to Create AdditionalV_(H)/V_(L) Interface Modifications that Discriminate from the NativeImmunoglobulin V_(H)/V_(L) Interface

Using PDB ID 3TCL (see, McLellan et al. (2011), Nature 480(7377);336-343) and a multi-state design protocol essentially as described inExample 1, additional compensatory V_(H)/V_(L) interface mutations thatsteer designed V_(H) and V_(L) domains from binding WT V_(H) and V_(L)domains are designed. Discrete designs are physically constructed withinthe pertuzumab IgG Fv region with different design paradigms (i.e.,mutations with very different amino acid combinations and residuepositions). The design constructs are mutated, expressed in HEK293Fcells, and tested for expression level and thermal stability usingsimilar protocols as described in Examples 1-3 above. Three discretedesigns as depicted in Table 9 (denoted H.4, H.5, and H.6) are shown toexpress well and are about as stable as WT pertuzumab.

TABLE 9 Sequence ID numbers and thermal challenge data for Designs H.4,H.5, and H.6. HC SEQ LC SEQ Construct Mutations ID NO: ID NO: T₅₀ (° C.)Pertuzumab None 1 2 61.8 ± 0.5  H.4 LC_Q38R, 29 30 60.3 ± 0.5  HC_Q39YH.5 LC_Q38R, 31 30 59.2 ± 0.5  HC_Q39F H.6 LC_Q38R, 32 30 60.6 ± 0.5 HC_Q39W

Example 5. Combining the C_(H)1/C_(L) and V_(H)/V_(L) Interface DesignsResults in Highly Specific HC/LC Pairing

In this example, the variable domain Designs H.4, H.6 and AB areexamined along with the constant domain Designs 2.1.3.2 and 2.1.3.3a.The specificity afforded by the V_(H)/V_(L) and C_(H)1/C_(L) designs inisolation and in combination with each other is measured using a similarLC/MS competition experiment as described in Example 3 (with theIgG/C_(L) proteins lacking variable domains), but here full-lengthimmunoglobulin heavy chains and light chains are used. In theexperiment, two full-length light chains (with V_(L) and C_(L)) areco-expressed and forced to compete for binding to a single heavy chainprior to secretion. Identities of the designs and the corresponding SEQID numbers for the constructs used in the specificity experiments areprovided in Table 10. Again, the pertuzumab V_(H) and V_(L) sequenceswere used in every construct as a vector for the designs.

TABLE 10 Identity and Sequence identification for constructs used inExample 5. HC SEQ LC SEQ HC Name^(a,b) ID NO: LC Name^(a,b) ID NO: WT +WT HC 1 WT + WTλ LC 2 AB + WT HC 25 AB + WTλ LC 26 H.4 + WT HC 29 WT +WTκ LC 33 H.6 + WT HC 32 AB + WTκ LC 34 WT + 2133a HC 35 H.4 + WTλ LC 30AB + 2133a HC 37 H.6 + WTλ LC 30 AB + 2132 HC 37 WT + 2133aλ LC 36 H.4 +2133a HC 39 AB + 2133aλ LC 38 H.4 + 2133aλ LC 40 AB + 2132λ LC 42 H.4 +WTκ LC 41 ^(a)Nomenclature is provided by having the first twocharacters of each protein specifying the variable domain design, whilethe subsequent numbering specifies the constant domain design (e.g.,‘WT + WTκ LC’ is a LC with WT pertuzumab V_(L) and WT kappa C_(L), while‘AB + 2133aλ LC’ is a LC with Design AB in its V_(L) and Design 2.1.3.3ain its lambda C_(L). Similarly, ‘AB + 2133a HC’ is a HC with Design ABin its V_(H) and Design 2.1.3.3a in its C_(HL).) ^(b)Some designs onlyhave sequence differences in their HC or LC (e.g., AB + 2133a and AB +2132 share identical HCs). The LC/MS specificity data for thecombinations tested is provided in Table 11.

TABLE 11 LC/MS analysis of the V_(H)/V_(L) and C_(H)1/C_(L) interfacedesigns in isolation and in combination. Ex- pres- % % sion Assembly^(a)Assembly^(a) (μg/ LC1 LC2 HC (LC1/HC) (LC2/HC) mL) WT + WTλ WT + WTκWT + WT 18 82 69 LC LC HC AB + WTλ WT + WTκ WT + WT 10 90 73 LC LC LCAB + WTλ WT + WTκ AB + WT 61 39 108 LC LC HC AB + WTκ WT + WTλ WT + WT40 60 132 LC LC HC AB + WTκ WT + WTλ AB + WT 39 61 112 LC LC HC H.4 +WTλ WT + WTκ WT + WT 39 61 94 LC LC HC H.4 + WTλ WT + WTκ H.4 + WT 54 44105 LC LC HC H.6 + WTλ WT + WTκ WT + WT 48 52 90 LC LC HC H.6 + WTλ WT +WTκ H.6 + WT 54 46 100 LC LC HC H.4 + WTλ AB + WTκ AB + WT 23 77 95 LCLC HC H.4 + WTλ AB + WTκ H.4 + WT 71 29 71 LC LC HC H.6 + WTλ AB + WTκAB + WT 25 75 67 LC LC HC H.6 + WTλ AB + WTκ H.6 + WT 69 31 94 LC LC HCWT + 2133aλ WT + WTλ WT + WT 50 50 29 LC LC HC WT + 2133aλ WT + WTλ WT +2133a 79 21 28 LC LC HC WT + 2133aλ WT + WTκ WT + WT 78 22 31 LC LC HCWT + 2133aλ WT + WTκ WT + 2133a 79 21 19 LC LC HC AB + 2133aλ WT + WTλWT + WT 42 58 51 LC LC HC AB + 2133aλ WT + WTλ AB + 2133a 85 15 87 LC LCHC H.4 + 2133aλ WT + WTλ WT + WT 47 53 61 LC LC HC H.4 + 2133aλ WT + WTλH.4 + 2133a 77 23 70 LC LC HC AB + 2132λ H.4 + WTκ H.4 + WT 11 89 66 LCLC HC AB + 2132λ H.4 + WTκ AB + 2132 74 26 98 LC LC HC AB + 2133aλ H.4 +WTk H.4 + WT 15 85 74 LC LC HC AB + 2133aλ H.4 + WTk AB + 2133a 78 22121 LC LC HC AB + 2132λ H.4 + WTλ H.4 + WT 9 91 94 LC LC HC AB + 2132λH.4 + WTλ AB + 2132 80 20 110 LC LC HC AB + 2133aλ H.4 + WTλ H.4 + WT 9± 1 91 ± 1  76 ± 3  LC LC HC AB + 2133aλ H.4 + WTλ AB + 2133a 87 ± 2  13± 3  70 ± 5  LC LC HC ^(a)The percent assembly is calculated based onthe relative area under the deconvoluted mass spectrometry peaks (i.e.,proportional to the number of counts hitting the detector) of each ofthe LCs co-purified bound to the HC prior to mass spectrometry analysis.Purified samples are reduced with DTT prior to analysis-as described inExample 2.

The data in Table 11 indicates that generating specificity at theV_(H)/V_(L) interface by combining Design AB in one Fab and Design H.4in the other Fab resulted in significant specificity (˜70-80%specificity) without any constant domain designs. Pairing the mostselectively specific V_(H)/V_(L) domain designs (AB in one Fab and H.4in the other) with the most selective constant domain designs (2.1.3.2or 2.1.3.3a) resulted in improved specificity overall, indicating thatwhile the variable domains may dominate, the constant domains contributeto specificity (Table 11). The best combination used a combination ofDesign AB (in V_(H)/V_(L)) and 2.1.3.3a (in C_(H)1/C_(L)) in Fab #1 andDesign H.4 (in V_(H)/V_(L)) with a WT C_(H)1/C_(L) in Fab #2. Thiscombination repeatedly generated a highly specific set of HC/LCinteractions with roughly 90% specificity in both directions (Table 11).

Example 6. Achieving Improved Specific HC/LC Fab Assembly within IgGBispecific Antibodies (BsAbs)

Two sets of IgG bispecific antibodies (BsAbs) are constructed to testhow combining the designs AB2133a and H.4WT may enable specific heavychain/light chain assembly of specific Fabs. All subcloning andmutagenesis protocols followed are essentially as described in previousExamples. The first BsAb consists of a combination of pertuzumab(anti-HER-2) and matuzumab (anti-EGFR) (see, Bier et al. (1998), CancerImmunol. Immunother. 46; 167-173) and the second consists of acombination of MetMAb (anti-cMET) (see, Jin et al. (2008), Cancer Res.68; 4360-4368) and an anti-Axl antibody YW327.6S2 (see, WO2011/014457and Ye et al. (2010), Oncogene 29; 5254-5264). All sequences for thenative antibodies are publicly available. To simplify our ability toobserve specific assembly using LC/MS, all HCs are deglycosylated bymutating asparagine 297 (the site of N-linked glycosylation in theantibody C_(H)2 domain) to glutamine. To promote heterodimerization inthe IgG-Fc, aspartic acid 399 and glutamic acid 356 are both mutated tolysine in one of the heavy chains of the anti-HER-2/anti-EGFR pair andone of the heavy chains of the anti-MET/anti-Axl pair. The remainingheavy chain in each pair had lysine 409 and lysine 392 mutated toaspartic acid (see, Gunasekaran et al. (2010), JBC 285; 19637-19646). Itshould be noted that other designs to promote heavy chainheterodimerization may be substituted to achieve the same overallaffect. Sequence ID numbers of the immunoglobulin chains used togenerate the IgG BsAbs are provided in Table 12.

TABLE 12 Sequence ID numbers of the HCs and LCs constructed todemonstrate the specific assembly of IgG BsAbs using the design FabV_(H)/V_(L) and C_(H)1/C_(L) interfaces. SEQ SEQ ID ID HC Name NO: LCName NO: WT antibody sequences pG1 (pertuzumab) 1 pλ (pertuzumab V_(L)and C_(λ)) 2 pκ (pertuzumab V_(L) and C_(κ)) 33 mG1 (matuzumab) 43 mλ(matuzumab V_(L) and C_(λ)) 44 mκ (matuzumab V_(L) and C_(κ)) 45 METG1(METMAb) 46 METλ (METMAb V_(L) and C_(λ)) 47 METκ (METMAb V_(L) andC_(κ)) 48 AxlG1 (anti-Axl) 49 Axlλ (anti-Axl V_(L) and C_(λ)) 50 Axlκ(anti-Axl V_(L) and C_(κ)) 51 Control IgG HCs and LCs with WT (native)V_(H)/V_(L) and C_(H)1/C_(L) interfaces^(a) (−)pG1 52 pλ (pertuzumabV_(L) and C_(λ)) 2 (+)mG1 53 mλ (matuzumab V_(L) and C_(λ)) 44 (−)METG154 mκ (matuzumab V_(L) and C_(κ)) 45 (+)AxlG1 55 METλ (METMAb V_(L) andC_(λ)) 47 Axlλ (anti-Axl V_(L) and C_(λ)) 50 Axlκ (anti-Axl V_(L) andC_(κ)) 51 IgG HCs and LCs with design V_(H)/V_(L) and C_(H)1/C_(L)interfaces^(a,b) AB + 2133a(−)[pG1] 13 AB + 2133a[pλ] 38 H.4 +WT(+)[mG1] 57 AB + 2133a[pκ] 56 AB + 2133a(−)[METG1] 60 H.4 + WT[mλ] 58H.4 + WT(+)[AxlG1] 17 H.4 + WT[mκ] 59 AB + 2133a[METλ] 61 AB +2133a[METκ] 62 H.4 + WT[Axlλ] 16 H.4 + WT[Axlk] 14 ^(a)The HC designswith (−) contained the K409D and K392D mutations while the HC designswith the (+) contained the D399K and E356K mutations. Both the (+) and(−) - containing HCs also have the N297Q mutation to eliminate N-linkedglycosylation. ^(b)Nomenclature is essentially as described in Table 10above. The first two characters of each protein specify the variabledomain design, while the subsequent numbering specifies the constantdomain design (e.g., a LC designated ‘AB + 2133a[pλ]’ contains Design ABin its V_(L) and Design 2.1.3.3a in its lambda C_(L). Similarly, an HCdesignated ‘AB + 2133a(−)[pG1]’ contains Design AB in its V_(H) andDesign 2.1.3.3a in its C_(HL), and the K409D and K392D substitutions inthe CH3 domain.) The notation appearing within the brackets refers tothe variable and constant domain of the particular parental antibody HCor LC containing the indicated design (e.g., “[pG1]” refers to a heavychain containing a pertuzumab variable domain and IgG1 constant domain,whereas “[mG1]” refers to a heavy chain with a matuzumab variable domainand IgG1 constant domain; similarly, “[pλ] refers to a light chain withpertuzumab variable domain and λ constant domain, whereas “[METλ]”refers to a light chain with METMAb variable domain and λ constantdomain)

To determine if the designed V_(H)/V_(L) (AB and H.4) and C_(H)1/C_(L)(2.1.3.3a and WT lamda or kappa) interfaces would enable specific IgGBsAb assembly over what occurs naturally with no FAb interface designsin place, transient transfections of particular designed Fab constructsfollowed by LC/MS analyses are performed. For each IgG BsAb, two heavychains and two light chains are simultaneously transfected usingseparate plasmids into mammalian HEK293F cells using transfectionprotocols essentially as described in the previous Examples. Thedesigned IgG BsAbs include the deglycosylation mutation and Fcheterodimerization mutations as described above. Further, a control setof IgG BsAbs with the deglycosylation and Fc heterodimerizationmutations and light chains with both C_(κ) or C_(λ) domains, but withoutthe designed V_(H)/V_(L) and C_(H)1/C_(L) modifications, are alsocreated by transfection of appropriate heavy chains and light chains. Ineach Designed IgG BsAb pair, one heavy chain and light chain (shownviable in both the C_(λ) and C_(κ) isotype) contained Design H.4, whilethe other heavy chain and light chain (shown viable in both the C_(λ)and C_(κ) isotype) contained Design AB and Design 2.1.3.3a. The exactheavy chain and light chain composition of each IgG BsAb synthesized isprovided in Table 13.

TABLE 13 The HC and LC elements of each IgG BsAb and the resultingpercentage of correct and incorrect IgG BsAb assembly based on the LC/MSintensities of the fully heterotetrameric species. Anti-HER-2/Anit-EGFRIgG BsAbs % % % LC1LC2^(a) LC1₂ LC2₂ IgG BsAb^(b) HC1^(c) LC1^(c)HC2^(c) LC2^(c) (correct) (incorr.) (incorr.) HEControl (−)pG1 pλ (+)mG1mλ 65 23 12 λλ^(c) HEControl (−)pG1 pλ (+)mG1 mκ 75 0 15 λκ HEDesignAB + 2133a AB + 2133a H.4 + WT H.4 + WT 90 8 2 λλ (−)[pG1] [pλ] (+)[mG1][mλ] HEDesign AB + 2133a AB + 2133a H.4 + WT H.4 + WT 90 7 3 λκ (−)[pG1][pλ] (+)[mG1] [mκ] HEDesign AB + 2133a AB + 2133a H.4 + WT H.4 + WT 8218 0 κλ (−)[pG1] [pκ] (+)[mG1] [mλ] HEDesign AB + 2133a AB + 2133a H.4 +WT H.4 + WT 87 10 3 κκ (−)[pG1] [pκ] (+)[mG1] [mλ] Anti-cMET/Anti-AxlIgG BsAbs % % % LC1LC^(a) LC1₂ ^(a) LC2₂ ^(a) IgG BsAb HC1^(c) LC1^(c)HC2^(c) LC2^(c) (correct) (incorr) (incorr) MAControl (−)METG1 METλ(+)AxlG1 Axlλ 70 27 3 λλ MAControl (−)METG1 METλ (+)AxlG1 Axlκ 61 38 1λκ MADesign AB + 2133a AB + 2133a H.4 + WT H.4 + WT 95 5 0 λλ (−)[METG1][METλ] (+)[AxlG1] [Axlλ] MADesign AB + 2133a AB + 2133a H.4 + WT H.4 +WT 100 0 0 λκ (−)[METG1] [METλ] (+)[AxlG1] [Axlκ] MADesign AB + 2133aAB + 2133a H.4 + WT H.4 + WT 97 2 1 κλ (−)[METG1] [METκ] (+)[AxlG1][Axlλ] MADesign AB + 2133a AB + 2133a H.4 + WT H.4 + WT 90 7 3 κκ(−)[METG1] [METκ] (+)[AxlG1] [Axlλ] ^(a)The LC/MS method is sensitivefor heterotetrameric IgGs containing mismatched HC pairs (HC1HC1 orHC2HC2), but none were detected. The percent values represent therelative counts detected for covalently linked (non-reduced)heterotetramers HC1HC2LC1LC2 (correctly formed) compared to incorrectHC1HC2LC1LC1 (incorr.) and incorrect HC1HC2LC2LC2 (incorr.). ^(b)EachBsAb is designated λλ, λκ, κλ, or κκ based on the C_(L) compositions(lambda or kappa) of its LCs. ^(c)Nomenclature for each heavy chain orlight chain construct is essentially as described above in Tables 10 and12

The data in Table 13 demonstrates that incorporating the V_(H)/V_(L) andC_(H)1/C_(L) designs into the IgG BsAbs significantly improved thecorrect assembly of the desired heterotetrameric species (i.e.,HC1/LC1+HC2/LC2). Without the Fab designs, the average correct assemblywas about 70% and 65% for the anti-HER-2/anti-EGFR andanti-cMET/anti-Axl IgG BsAbs, respectively. With the Fab designsincorporated, the average correct assembly was about 87% and 96% for theanti-HER-2/anti-EGFR and anti-cMET/anti-Axl IgG BsAbs, respectively.

Next, the IgG BsAbs may be tested for their oligomeric nature usinganalytical size exclusion chromatography (SEC). First, the IgG BsAbs arepurified at the 1 mL scale from HEK293F supernatants using the protein Gmagnetic bead procedure, essentially as described in Example 1. For SECanalysis, between 10-50 μg of each protein was applied to a Yarra G3000(7.8×300 mm) analytical SEC column (Phenomenex) with all other assayparameters similar to the protocol as described in Example 1. SEC of thepertuzumab (anti-HER-2) and matuzumab (anti-EGFR) control IgGsdemonstrated slightly different SEC retention times for the two proteinsdue to differences in their variable domains. Further, theanti-HER-2/anti-EGFR IgG BsAbs tested demonstrate primarily monomericbehavior with SEC retention times between what was observed fornon-bispecific pertuzumab and matuzumab controls, which might beexpected if the proteins contain one pertuzumab Fab and one matuzumabFab. Similar to pertuzumab and matuzumab, the METMAb and anti-Axlcontrol IgGs demonstrate slightly different retention times by SEC. Theanti-cMet/anti-Axl IgG BsAbs also demonstrate primarily monomericbehavior with retention times approximating the average of the controlantibodies, non-bispecific antibodies. In addition, the two control IgGanti-cMet/anti-Axl BsAbs that did not have the Fab specificity designsincorporated, and which demonstrated significant populations ofmismatched light chain pairings, also showed multiple monomeric speciesby SEC. The four anti-cMet/anti-Axl BsAbs containing the Fab specificitydesigns did not demonstrate this behavior.

Example 7 Functional Activity of Designed IgG BSAbs

The dual-binding behavior of the synthesized BsAbs may be assessed usingboth sandwich ELISA and surface plasmon resonance assays as follows.

Two sandwich ELISAs are developed, one for detectinganti-HER-2/anti-EGFR BsAb activity and one for detectinganti-cMET/anti-Axl BsAb activity. For both ELISAs, clear 96-well roundbottom high binding Immulon microtiter plates (Greiner bio-one; cat#650061) are coated overnight at 2-8° C. with 50 μL/well 1 μg/mLhHER-2-Fc or 1 μg/mL hHGFR(cMet)-Fc (both from R&D systems) in a 50 mMNa₂CO₃ pH 8 buffer. The plates are washed 4 times with PBST and blockedwith 100 μL/well casein buffer (Pierce) for 1 hr at 37° C. The platesare then washed 4 times with PBST and the parental IgG controls or BsAbIgG test articles are added at 50 μL/well and 5 μg/mL and seriallydiluted 1:3 down the plate. The test articles are incubated on the platefor 1 hr at 37° C. The plates are then washed 4 times with PBST and 50μL/well 0.2 μg/mL hEGFR-Fc-biotin or hAxl-Fc-biotin (both from R&Dsystems) is added for 1 hr at 37° C. The plates are then washed 4 timeswith PBST followed by the addition of a 50 μL/well streptavidin-HRP(Jackson Labs) diluted 1:2000 in PBST. The streptavidin-HRP is incubatedin each well for 1 hr at 37° C. The plates are then washed 4 times withPBST and 100 μL/well 1-component TMB substrate is added (KPLlaboratories). After approximately 10 minutes, 100 mL/well 1% H₃PO₄ (inH₂O) is added to each plate to quench the reaction. Absorbance (450 nm)of every well in the plates is read using a SpectraMax UV plate reader(Molecular Devices). Biotin-labeling of the hEGFR-Fc and hAxl-Fcproteins is performed using EZ-Link Sulfo-NHS-LC-Biotin (ThermoScientific) according to the manufacturer's protocol.

Both sandwich ELISAs were capable of demonstrating the dual-bindingbehavior of the BsAbs. For both the anti-HER-2/anti-EGFR and theanti-cMet/anti-Axl IgG BsAbs, the monoclonal IgG controls (pG1, mG1,METG1, or AxlG1) demonstrated no ability to generate signal in thesandwich ELISAs. The control IgG BsAbs with no Fab redesigns(HEControlλλ, HEControlλκ, MAControlλλ, MAControlλκ) generated sigmoidalbinding curves in the assay. All the IgG BsAbs with the Fab redesignsalso demonstrated sigmoidal binding curves. Average EC₅₀ values for thecontrol and designed IgG BsAbs were as follows: HEControl EC₅₀=470±90ng,/mL; HEDesign EC₅₀=280±20 ng/mL; MAControl EC₅₀=260±80; and MADesignEC₅₀=220±20.

Surface plasmon resonance experiments may also be performed to evaluatethe dual-binding behavior of the BsAbs using, for example, on aBiacore3000 using HBS-EP as the running buffer (GE Healthcare). Briefly,hHER-2-Fc-biotin and hAxl-Fc-biotin (both at 20 μg/mL) are immobilizedonto SA sensorchip surfaces by injection of 20 over 2 minutes at 10μL/min. Next, 20 μL of each control IgG, control IgG BsAb, or designedIgG BsAb, diluted to 30 nM in HBS-EP buffer, is injected over thesesensorchip surfaces at 5 μL/min followed by a secondary 20 μL injectionof 20 nM hEGFR-Fc or hHGFR(cMet)-Fc. The hHER-2-Fc and hAxl-Fcsensorchip surfaces are regenerated by raising the flow rate to 50μL/min and injecting two 5 μL injections of a 0.1 M glycine solution atpH 2.5 and pH 2.0, respectively.

None of the monoclonal IgGs (pG1, mG1, METG1, or AxlG1) demonstrateddual binding activity in the assay (FIGS. 2 and 3). Both the control IgGBsAbs and the BsAbs containing Fab designs demonstrated strongdual-binding activity in the assay.

Example 8—Optimization of Constant Domain Design 2133a Using CH1/Ckappa

In certain contexts, constant domain (C_(u)1/C_(L)) design denoted 2133awas determined to provide less correct HC/LC pairing specificity whenused in C_(H)1/C_(κ) compared to its use in C_(H)1/C_(λ) (See, forexample the EGFR×HER2 bispecific antibody assembly in Table 13). It wasalso found that the 2133a constant domain design destabilized when usedin C_(H)1/C_(κ) compared to WT C_(H)1/C_(κ) (see Table 15, below).

To assess stability of the 2133a design IgG1/C_(κ) proteins lackingvariable domains [WT=SEQ ID NO: 3 (HC) and 63 (LC); Design 2133a=SEQ IDNO: 18 (HC) and 64 (LC)] are generated using molecular biology,expression, and purification methods as generally described in previousexamples. The stability of the two purified proteins (WT and 2133a IgGslacking variable domains or Fvs) are characterized using differentialscanning calorimetry (DSC) as follows. The midpoints of the thermalunfolding transitions (denoted ‘T_(m)’) of the C_(H)1/C_(κ) domainsprovide a measure of their relative stability. The T_(m) of the2133a-containing C_(H)1/C_(κ) domain was 67.7° C. while that of WTC_(H)1/C_(κ) was 70.8° C. DSC is performed using an automated capillaryDSC (capDSC, GE Healthcare). Protein solutions and reference (buffer)solutions are sampled automatically from a 96-well plate using, therobotic attachment. Before each protein scan, at least one buffer/bufferscan is performed, to define the baseline for subtraction. All 96-wellplates containing protein are stored within the instrument at 6° C.Samples are run at 1.0 mg/ml protein concentration in PBS. Scans areperformed from 10 to 95° C. at 90° C./hr using the low feedback mode.Scans are analyzed using the origin software supplied by themanufacturer. Subsequent to the subtraction of reference baseline scans;nonzero protein scan baselines are corrected using a third-orderpolynomial. Based on the analyses, the IgG1/C_(κ) protein harboring the2133a design was less stable than the WT protein (see Table 15, below).

Utilizing an in-house crystal structure of C_(H)1/C_(λ) with the 2133adesign, we identified C_(H)1_A172 (design 2133a already contains anH172A substitution) and C_(H)1_V190 as residue positions to randomize.The 2133a IgG1/C_(κ) constructs lacking variable domains (SEQ ID NO: 18and 64) are used to create the libraries for screening and analysis. Thelibraries are generated using the QuikChange II Site-DirectedMutagenesis Kit (Agilent) using protocols provided by the manufacturer.The constructs are expressed via transient transfection in HEK293F cellsas generally described in Example 1. Titers of the proteins are assessedusing the HPLC Protein G quantitation and collection method as generallydescribed in Example 2. The proteins are assessed for their stabilityproperties using a thermal challenge assay similar to that described inExample 1. Unique to this assay, the plates are coated with a sheepanti-human Fd (CH1) polyclonal antibody (Meridian Life Sciences, cat#W90075C) at 1 μg/ml, and 100 μL/well in a 0.05 M NaHCO₃ buffer, pH 8.3for 1 hr at 37° C. or overnight at 4° C. Thermally resistant IgG1/C_(κ)protein is detected by adding a detection antibody (HRP-labeled goatanti-human kappa, Southern Biotechnology, cat #2060-5) at a 1:10,000dilution in PBS-T into every well at 100 μL/well and incubating for 1 hrat 37° C. Other assay parameters are generally as described previously.

From the library, three HC mutants, A172R, V190M, and V190I, areidentified that stabilized the 2133a-containing C_(H)1/C_(κ) domains inthe thermal challenge assay. Combinations comprising C_(H)1_A172R_V190Mor C_(H)1_A172R_V190I were also generated. Table 14 provides a listingof Sequence ID numbers for these 2133a C_(H)1 mutant proteins.

Larger scale (≥100 mL) transfections of the single and double mutantmodifications of design 2133a C_(H)1/C_(κ) constructs are generated inHEK293F cells. The transfected cells are cultured as generally describedin Example 1 for small scale cultures. Supernatants with protein areclarified using 0.2 μm filters. The C_(H)1/C_(κ) (−Fv) proteins arepurified using standard protein A affinity chromatography methods. Theproteins are buffer exchanged into PBS and analyzed by DSC as describedabove for the WT and 2133a C_(H)1/C_(κ) (−Fv) proteins. The results ofthe DSC analyses showed that the single and double mutant combinationswere stabilizing to the 2133a C_(H)1/C_(κ) domains (Table 15).

TABLE 14 Sequence ID numbers of the HC and LC designs improving thestability or specificity of the 2133a design in CH1/Cκ. SequenceModified 2133a HC for Improved Stability ID number HC (−V_(H))2133a_A172R SEQ ID 65 HC (−V_(H)) 2133a_V190M SEQ ID 66 HC (−V_(H))2133a_V190I SEQ ID 67 HC (−V_(H)) 2133a_A172R_V190M SEQ ID 68 HC(−V_(H)) 2133a_A172R_V190I SEQ ID 69 Cκ_2133a_V133L SEQ ID 70Cκ_2133a_S174Q SEQ ID 71 Cκ_2133a_S174D SEQ ID 72 Cκ_2133a_V133L_S174QSEQ ID 73 Cκ_2133a_V133L_S174D SEQ ID 74 (−)PG1_AB2133a^(a) SEQ ID 13(−)PG1_AB2133a_MR^(b) SEQ ID 75 (−)PG1_AB2133a_IR^(b) SEQ ID 76(−)MetG1_AB2133a SEQ ID 60 (−)MetG1_AB2133a_MR^(b) SEQ ID 77(−)MetG1_AB2133a_IR^(b) SEQ ID 78 Pκ_AB2133a SEQ ID 56 Pκ_AB2133a_LD^(b)SEQ ID 79 Pκ_AB2133a_LQ^(b) SEQ ID 80 Metκ_AB2133a SEQ ID 62Metκ_AB2133a_LD^(b) SEQ ID 81 Metκ_AB2133a LQ^(b) SEQ ID 82(+)MG1_H4^(a) SEQ ID 57 (+)TG1_H4^(a) SEQ ID 83 Mκ_H4 SEQ ID 59 Tκ_H4SEQ ID 87 ^(a)The HC designs with (−) contained the K409D and K392Dmutations while the HC designs with the (+) contained the D399K andE356K mutations. Both the (+) and (−) − containing HCs also have theN297Q mutation to eliminate N-linked glycosylation. ^(b)‘MR’ refers toC_(H)1 mutations A172R and V190M. ‘IR’ refers to C_(H)1 mutations A172Rand V190L ‘LD’ refers to Cκ mutations V133L and S174D. ‘LQ’ refers to Cκmutations V133L and S174Q.

TABLE 15 Expression and differential scanning calorimetry (DSC) resultsfor mutants of the 2133a-containing IgG1/Cκ protein lacking variabledomains. Expression DSC T_(m) Protein Level (μg/mL) (° C.)^(a) HC(−V_(H)) WT/Cκ_WT 15 70.8 ± 0.1  HC (−V_(H)) 2133a/Cκ_2133a 19 67.7 ±0.2  HC (−V_(H)) 2133a_A172R/Cκ_2133a 16 70.0 HC (−V_(H))2133a_V190M/Cκ_2133a 21 69.1 HC (−V_(H)) 2133a_V190I/Cκ_2133a 14 68.6 HC(−V_(H)) 2133a_A172R_V190M/Cκ_2133a 43 70.1 HC (−V_(H))2133a_A172R_V190I/Cκ_2133a 43 69.2 ^(a)The DSC Tm refers to the midpointof the cooperative unfolding transition of the C_(H)1/Cκ heterodimer ineach of these constructs.

Using the LC competition assay as generally described in Example 2, thespecificity afforded by the 2133a design mutations in C_(H)1/C_(κ)instead of C_(H)1/C_(λ) is assessed. As shown in Table 16 below, the WTC_(κ) protein (SEQ ID 63) shows little tendency to associate with the2133a HC (SEQ ID 18) protein with or without the stabilizing mutationsdepicted in Table 15. However, it was observed that the 2133a-containingC_(κ) domain (SEQ ID 64) could associate with a WT HC (SEQ ID 3) morestrongly than a WT C_(κ) domain (SEQ ID 63, see Table 16, below).

Using in-house 2133a/2133a and WT/WT C_(H)1/C_(λ) crystal structures,2133a C_(κ) residue positions V133 and S174 were identified as positionswhere potential new interactions with 2133a-containing C_(H)1 domainscould be generated, that might be incompatible with WT C_(H)1. Screeningfor 2133a C_(κ) mutations that maintained binding to the 2133a designHC, while decreasing binding with WT HC, three mutations (C_(κ_)V133L,C_(κ_)S174Q, and C_(κ_)S174D) were found to provide beneficialspecificity in the LC C_(κ) competition assay (Table 16) (Sequence IDnumbers of these constructs are provided in Table 14, above). Each ofthese mutations maintained the dominance of design 2133a C_(κ) bindingto 2133a C_(H)1 over WT C_(κ), while decreasing the proportion of design2133a C_(κ) binding to WT C_(H)1 compared to WT C_(κ). Further, theV133L mutation could be combined with either S174Q or S174D to providefurther improvements in specificity (Table 16).

TABLE 16 Competition of Ckappa proteins binding to a single IgG1 HC (novariable domains) with variants of the 2.1.3.3a design % Assembly^(a) %Assembly^(a) LC1 LC2 HC (LC1/HC) (LC2/HC) Cκ_2133a Cκ_WT HC (−V_(H)) WT86.6 ± 9.9  13.4 ± 9.9  Cκ_2133a Cκ_WT HC (−V_(H)) 2133a 98.8 ± 1.2  1.2± 1.2 Cκ_2133a_V133L Cκ_WT HC (−V_(H)) WT 70 30 Cκ_2133a_S174Q Cκ_WT HC(−V_(H)) WT 59 41 Cκ_2133a_S174D Cκ_WT HC (−V_(H)) WT 62 38Cκ_2133a_V133L_S174Q Cκ_WT HC (−V_(H)) WT 0 100 Cκ_2133a_V133L_S174QCκ_WT HC (−V_(H)) 2133a 100 0 Cκ_2133a_V133L_S174Q Cκ_WT HC (−V_(H))2133a 100 0 A172R_V190M Cκ_2133a_V133L_S174Q Cκ_WT HC (−V_(H)) 2133a97.8 2.2 A172R_V190I Cκ_2133a_V133L_S174D Cκ_WT HC (−V_(H)) WT 28.5 71.5Cκ_2133a_V133L_S174D Cκ_WT HC (−V_(H)) 2133a 100 0 Cκ_2133a_V133L_S174DCκ_WT HC (−V_(H)) 100 0 2133a_A172R_V190M Cκ_2133a_V133L_S174D Cκ_WT HC(−V_(H)) 100 0 2133a_A172R_V190I ^(a)As described in previous examples,the % assembly was derived using the ratio of counts for each LCdetected by the mass spectrometer after purifying the expressed IgG(−Fv)proteins using the anti-Fc, releasing the Cκ domains using DTT.

The 2133a C_(H)1 and Cκ mutants were then added to two separatefull-length HCs and LCs (including variable domains) to assess theirability to impact specific bispecific antibody assembly. The 2133amutants A172R and V190M (denoted ‘MR’) and A172R and V190I (denoted‘IR’) were each combined for the testing as were the compatible design2133a Cκ mutants V133L and S174D (denoted ‘LD’) and V133L and S174Q(denoted ‘LQ’). The mutants were added to both a Pertuzumab (anti-HER2)and MetMAb (anti-cMet) IgG1/kappa HC/LC pair containing the 2133adesigns. To promote heavy chain heterodimerization, the Pertuzumab andMetMAb HCs contained the K409D and K392D mutations, denoted ‘(−)’, whilethe complementary Matuzumab and Trastuzumab HCs contained the D399K andE356K mutations, denoted ‘(+)’ (see, Gunasckaran et al., (2010), JBC285; 19637-19646). Both the (+) and (−)-containing HCs also have theN297Q mutation to eliminate N-linked glycosylation. The Pertuzumab HC/LCpairs were co-expressed with an H4WT-containing Matuzumab IgG1/kappaHC/LC pair to form a HER2×EGFR IgG bispecific antibody. The MetMAb HC/LCpairs were co-expressed with an H4WT-containing Trastuzumab IgG1/kappaHC/LC pair to form a cMet×HER2 IgG bispecific antibody. The Sequence IDnumbers for all sequences used in the study are provided in Table 14,above. All LCs in this study were fully kappa (i.e., VκCκ).

The ability to co-express each of the antibody pairs consisting of 2 HCsand 2 LCs (four chains total) and have them assemble into correctlyformed IgG BsAbs was determined by MS as generally described in Example6, above. Results of the co-expression studies are provided in Table 17,below. All data points are the average of between 3 and 9 individualmeasurements and the error represents the standard deviation.Differences between the correct assembly in the absence and presence ofthe ‘MR’, ‘IR’, ‘LD’, and/or ‘LQ’ designs were tested for theirsignificance using a standard or paired t-test.

TABLE 17 Specific Assembly of IgG BsAbs Using 2133a Variant CH1/CkappaDesigns. P-value % Correct (One- IgG BsAb tailed HC1^(a,b) LC1^(a)HC2^(a,b) LC2^(a) w/Std.Dev. t-test) (−)PG1_AB2133a Pκ_AB2133a (+)MG1_H4Mκ_H4 71.8 ± 4.5  (n = 9) (−)PG1_AB2133a_MR Pκ_AB2133a (+)MG1_H4 Mκ_H462.2 ± 3.3  N/A (n = 5) (−)PG1_AB2133a_IR Pκ_AB2133a (+)MG1_H4 Mκ_H456.6 ± 2.4  N/A (n = 5) (−)PG1_AB2133a Pκ_AB2133a_LD (+)MG1_H4 Mκ_H476.3 ± 7.1  P = 0.063 (n = 9) (−)PG1_AB2133a Pκ_AB2133a_LQ (+)MG1_H4Mκ_H4 82.9 ± 13.1 P = 0.015 (n = 8) (−)PG1_AB2133a_MR Pκ_AB2133a_LD(+)MG1_H4 Mκ_H4 94.9 ± 1.7  P < 0.001 (n = 5) (−)PG1_AB2133a_MRPκ_AB2133a_LQ (+)MG1_H4 Mκ_H4 77.3 ± 1.7  P = 0.01 (n = 5)(−)PG1_AB2133a_IR Pκ_AB2133a_LD (+)MG1_H4 Mκ_H4 76.2 ± 9.6  P = 0.14 (n= 4) (−)PG1_AB2133a_IR Pκ_AB2133a_LQ (+)MG1_H4 Mκ_H4 91.2 ± 3.05 P <0.001 (n = 3) P-value (One- % Correct tailed IgG BsAb paired HC1^(a,b)LC1^(a) HC2^(a,b) LC2^(a) w/Std.Dev. t-test) (−)MetG1_AB2133aMetκ_AB2133a (+)TG1_H4 Tκ_H4 51.8 ± 2.8  (n = 3) (−)MetG1_AB2133a_MRMetκ_AB2133a (+)TG1_H4 Tκ_H4 44.9 ± 11.6 N/A (n = 3) (−)MetG1_AB2133a_IRMetκ_AB2133a (+)TG1_H4 Tκ_H4 34.8 ± 8.4  N/A (n = 3) (−)MetG1_AB2133aMetκ_AB2133a_LD (+)TG1_H4 Tκ_H4 73.7 ± 1.6  P = 0.006 (n = 3)(−)MetG1_AB2133a Metκ_AB2133a_LQ (+)TG1_H4 Tκ_H4 65.5 ± 2.1  P < 0.001(n = 3) (−)MetG1_AB2133a_MR Metκ_AB2133a_LD (+)TG1_H4 Tκ_H4 61.0 ± 3.1 P = 0.015 (n = 3) (−)MetG1_AB2133a_MR Metκ_AB2133a_LQ (+)TG1_H4 Tκ_H450.1 ± 1.  Not (n = 3) significant (−)MetG1_AB2133a_IR Metκ_AB2133a LD(+)TG1_H4 Tκ_H4 56.2 ± 1.6  P = 0.035 (n = 3) (−)MetG1_AB2133a_IRMetκ_AB2133a LQ (+)TG1_H4 Tκ_H4 50.7 ± 2.0  Not (n = 3) significant^(a)All Pertuzumab and MetMAb molecules have the AB2133a HC/LC designwith or without the further HC or LC mutations. All Matuzumab andTrastuzumab molecules have the H4 design mutations in their variabledomains. All LCs are fully Kappa (i.e., VκCκ). ^(b)The HC designs with(−) contained the K409D and K392D mutations while the HC designs withthe (+) contained the D399K and E356K mutations. Both the (+) and (−) -containing HCs also have the N297Q mutation to eliminate N-linkedglycosylation.

Both the ‘LD’ and ‘LQ’ paired mutations provided significantimprovements in correct LC pairing within the IgG bispecific antibodiesover utilization of unmodified AB2133a kappa LCs. Adding ‘LD’ and ‘LQ’mutant combinations to the AB2133a C_(κ) provided an average 13% and12%, respectively, benefit in correct LC assembly over what was obtainedin their absence. While the HC ‘MR’ and ‘IR’ mutations are stabilizingto the 2133a C_(H)1/C_(κ) (interaction, their impact on specificity wasless clear. Adding the HC ‘MR’ and ‘IR’ mutations in isolation decreasedthe correct IgG bispecific antibody assembly in all cases. In mostcases, adding the HC ‘MR’ or ‘IR’ mutants to either LC ‘LQ’ or ‘LD’mutations led to either no increase or a decrease in correct assembly;however, the addition of ‘IR’ to ‘LQ’ within the Anti-HER2×Anti-EGFR IgGbispecific antibody led to an approximate 10% increase in correctassembly over what was observed with ‘LQ’ alone. Thus, while the ‘MR’and ‘IR’ mutations improve the stability of design 2133aC_(H)1/C_(κ)-containing bispecific antibodies, it appears to be casespecific whether the ‘MR’ and ‘IR’ mutations improve specificity.

Example 9—Additional Specificity Designs to Improve LC Specificity whenExpressing IgG Bispecific Antibodies

To further improve the specificity of IgG bispecific antibody assembly,additional designs within the variable domains that drive improved HC/LCpairing specificity were pursued. A charge/polar pair of amino acids atHC_Q105/LC_K42 was identified for manipulation. LC_K42 was mutated to Dand HC_Q105 was mutated to R within the pertuzumab IgG1/kappa H4WTdesign. After analyzing expression and LC competition data (as generaldescribed in previous Examples), LC_K42D and HC_Q105R, when added to H4variable domain designs, provided slight improvement in H4(+K42D)pertuzumab LC pairing with its cognate HC H4(+Q105R) design whenco-expressed with an AB2133a pertuzumab LC (˜20% improvement).Co-expressing the H4(+K42D) pertuzumab LC with the design AB2133apertuzumab LC and HC also did not markedly decrease the observedassembly level of the design AB2133a pertuzumab HC and LC pair. Thisadditional design mutation (HC_Q105R/LC_K42D) is denoted as ‘DR’ in theTables 18 and 19 below.

Inspection of a Fab crystal structure (pdb id: 3HC4) reveals a lysine atHC residue 228 and an aspartic acid at LC residue 122 near the distalend of the antibody Fab located near to the HC/LC disulfide bond. Toprovide charge-based steering that may favor proper HC/LC pairing, thesecharged residues were modified (HC_K228D/LC_D122K) in Pertuzumab H4WTIgG1/kappa. (To ensure the HC/LC disulfide bond was not disrupted by thecharge swap, the HC residue 230 was mutated to glycine and HC residue127 was mutated to cysteine.) This charge swap and cysteine modificationis denoted ‘CS’.

To determine whether the ‘CS’ and ‘DR’ designs might improve correctHC/LC assembly when expressing IgG bispecific antibodies, these designswere tested in isolation and in combination in antibodies containing theH4WT IgG1/kappa design. The Pertuzumab IgG1/kappa molecules containingAB2133a designs were paired with other IgG1/kappa antibodies containingthe H4WT designs (with and without the ‘DR’ and/or ‘CS’ mutations)including BHA10 (Jordan, J L, et al. (2009) Proteins 77:832-41),Matuzumab, and Trastuzumab (i.e., Herceptin). Additionally, Pertuzumabwith H4WT IgG1/kappa (with and without the ‘DR’ and/or ‘CS’ mutations)was tested for bispecific IgG assembly with Trastuzumab-containing theAB2133a designs in IgG1/kappa. The Pertuzumab and Trastuzumab HCscontaining the AB2133a Fab designs contained the K409D and K392Dmutations, denoted ‘(−)’, while the complementary H4WT designs (with andwithout the ‘DR’ and/or ‘CS’ mutations) contained the D399K and E356Kmutations, denoted ‘(+)’ (see, Gunasekaran et al, (2010), JBC 285;19637-19646). Both the (+) and (−)-containing HCs also had the N297Qmutation to eliminate N-linked glycosylation. To generate IgG bispecificantibodies, the HC and LC from Pertuzumab are co-transfected with the HCand LC of a second antibody in 2 mL HEK293F cells. The resultingmaterial is secreted from the cells, purified as described in Example 2and characterized by MS. The Sequence ID numbers of all the HCs and LCsused in the experiment are provided in Table 18.

TABLE 18 Sequence ID numbers of the HC and LC designs improving thespecificity of HC/LC assembly. Sequence names for constructs used todemonstrate additional improvements in the specificity of Fab DesignsSEQ ID NO: (−)PG1 AB2133a SEQ ID 13 (+)PG1 H4WT SEQ ID 91 (+)PG1 H4WT +CS SEQ ID 92 (+)PG1 H4WT + DR SEQ ID 93 (+)PG1 H4WT + CS + DR SEQ ID 94Pκ AB2133a SEQ ID 56 Pκ H4WT SEQ ID 41 Pκ H4WT + CS SEQ ID 95 Pκ H4WT +DR SEQ ID 96 Pκ H4WT + CS + DR SEQ ID 97 (+)BHA10G1 H4WT SEQ ID 104(+)BHA10G1 H4WT + CS SEQ ID 105 (+)BHA10G1 H4WT + DR SEQ ID 106(+)BHA10G1 H4WT + CS + DR SEQ ID 107 (+)MG1 H4WT SEQ ID 57 (+)MG1 H4WT +CS SEQ ID 98 (+)MG1 H4WT + DR SEQ ID 99 (+)MG1 H4WT + CS + DR SEQ ID 100(+)TG1 H4WT SEQ ID 83 (+)TG1 H4WT + CS SEQ ID 84 (+)TG1 H4WT + DR SEQ ID85 (+)TG1 H4WT + CS + DR SEQ ID 86 (−)TG1 AB2133a SEQ ID 108 BHA10κ H4WTSEQ ID 109 BHA10κ H4WT + CS SEQ ID 110 BHA10κ H4WT + DR SEQ ID 111BHA10κ H4WT + CS + DR SEQ ID 112 Mκ H4WT SEQ ID 59 Mκ H4WT + CS SEQ ID101 Mκ H4WT + DR SEQ ID 102 Mκ H4WT + CS + DR SEQ ID 103 Tκ H4WT SEQ ID87 Tκ H4WT + CS SEQ ID 88 Tκ H4WT + DR SEQ ID 89 Tκ H4WT + CS + DR SEQID 90 Tκ AB2133a SEQ ID 113

The results of the MS measurements of percentage of correctly formedHC/LC pairs are provided in Table 19. All data points are the average ofbetween 3-5 individual measurements and the error represents thestandard deviation. Differences between the correct assembly in theabsence and presence of the ‘DR’, ‘CS’, or ‘CS+DR’ designs were testedfor their significance using a paired t-test.

TABLE 19 Specific assembly of IgG bispecific antibodies utilizing fullykappa LCs (i.e., VκCκ) with and without (i) VH_Q105R/VL_K42D (DR), (ii)CH1_S127C_K228D_C230G/CL_D122K (CS), or the combination of (i) and (ii).P-value (One- % Correct tailed IgG BsAb paired t- HC1^(a) LC1^(b)HC2^(a) LC2^(b) w/Std.Dev. test) Anti-HER2(pertuzumab)XAnti-LTbR(BHA10)IgG Coexpression (−)PG1 AB2133a Pκ AB2133a (+)BHA10G1 H4WT BHA10κ H4WT78.8 ± 6.6  (n = 3) (−)PG1 AB2133a Pκ AB2133a (+)BHA10G1 H4WT + CSBHA10κ H4WT + CS 87.2 ± 3.0  P = 0.03 (n = 3) (−)PG1 AB2133a Pκ AB2133a(+)BHA10G1 H4WT + DR BHA10κ H4WT + DR 84.7 ± 3.6  P = 0.07 (n = 3)(−)PG1 AB2133a Pκ AB2133a (+)BHA10G1 H4WT + CS + DR BHA10κ H4WT + CS +DR 87.4 ± 4.2  P = 0.03 (n = 3) Anti-HER2(pertuzumab) XAnti-EGFR(matuzumab) IgG Compression (−)PG1 AB2133a Pκ AB2133a (+)MG1H4WT Mκ H4WT 75.1 ± 6.1  (n = 3) (−)PG1 AB2133a Pκ AB2133a (+)MG1 H4WT +CS Mκ H4WT + CS 68.4 ± 4.0  Failed (n = 3) (−)PG1 AB2133a Pκ AB2133a(+)MG1 H4WT + DR Mκ H4WT + DR 88.9 ± 6.0  P = 0.06 (n = 3) (−)PG1AB2133a Pκ AB2133a (+)MG1 H4WT + CS + DR Mκ H4WT + CS + DR 84.4 ± 4.1  P= 0.02 (n = 3) Anti-HER2(pertuzumab) X Anti-HER2(trastuzumab) IgGCoexpression (−)PG1 AB2133a Pκ AB2133a (+)TG1 H4WT Tκ H4WT 69.7 ± 5.3 (n = 5) (−)PG1 AB2133a Pκ AB2133a (+)TG1 H4WT + CS Tκ H4WT + CS 71.7 ±6.7  P = 0.30 (n = 5) (−)PG1 AB2133a Pκ AB2133a (+)TG1 H4WT + DR TκH4WT + DR 70.2 ± 3.8  P = 0.28 (n = 5) (−)PG1 AB2133a Pκ AB2133a (+)TG1H4WT + CS + DR Tκ H4WT + CS + DR 79.6 ± 4.2  P < 0.0001 (n = 5)Anti-HER2(pertuzumab) X Anti-HER2(trastuzumab) IgG Coexpression withH4WT and AB2133a swapped (+)PG1 H4WT Pκ H4WT (−)TG1 AB2133a Tκ AB2133a35.4 ± 10.1 (n = 5) (+)PG1 H4WT + CS Pκ H4WT + CS (−)TG1 AB2133a TκAB2133a 41.7 ± 16.8 P = 0.07 (n = 5) (+)PG1 H4WT + DR Pκ H4WT + DR(−)TG1 AB2133a Tκ AB2133a 52.0 ± 9.0  P = 0.003 (n = 5) (+)PG1 H4WT +CS + Pκ H4WT + CS + DR (−)TG1 AB2133a Tκ AB2133a 47.6 ± 15.1 P = 0.01 DR(n = 5) ^(a)The HC designs with (−) contained the K409D and K392Dmutations while the HC designs with the (+) contained the D399K andE356K mutations. Both the (+) and (−) - containing HCs also have theN297Q mutation to eliminate N-linked glycosylation. ^(b)All light chainsin Table 19 were fully kappa (i.e., VκCκ).

As with the data in Example 8, it was clear from the data in Table 19that the AB2133a designs did not provide the same degree of specificitywhen using fully kappa LCs (VκCκ).

The data in Table 19 demonstrate trends showing that both the ‘DR’ and‘CS’ designs, when added to the H4WT-containing Fab, generally improvethe specificity of correct HC/LC pairing within IgG bispecificantibodies expressed in a single cell. In a few cases, the individual‘CS’ or ‘DR’ were found to significantly improve (i.e., P≤0.05) thecorrect assembly. However, when the two designs were combined (‘CS+DR’),the improvements in correct HC/LC pairing specificity were statisticallysignificant for all groups. Improvements in specificity were observedwhen the designs were placed on the H4WT-side of the IgG BsAb.

Overall the designs described in Example 8 and Example 9 can be utilizedto improve the HC/LC pairing stability and specificity within IgGbispecific antibodies. This was particularly evident when both LCs ofthe IgG bispecific antibody were fully kappa; a scenario where theAB2133a designs provide less specificity than when the AB2133a designsare used within a VκCλ LC.

Sequences: (mutations denoted by underlined, bold-face type)SEQ ID NO. 1: PERTUZUMAB HC (pG1)EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFILSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 2:PERTUZUMAB LC (pλ, KAPPA V_(L)/LAMBDA C_(L))DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 3:  HUMAN HC (MINUS VARIABLE DOMAINS) WILD-TYPE CONSTRUCTASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFPLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 4:  LAMBDA C_(L) DOMAINGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPTEC SEQ ID NO. 5: HC DESIGN 1.0 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFILSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHT T PAVLQSSGLYSLSS F VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 6:LC CHAIN DESIGN 1.0 DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVC F ISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 7:  HC minus V_(H) DESIGN 2.1, 2.1.2.1, 2.1.2.2ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG VHT GPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFPLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO. 8:C_(L) DESIGN 2.1, 2.1.1.1, AND 2.1.1.2 GQPKAAPSVTLFPPSSEELQANKATLVC AISDFYPGAVTVAWKADSSPV KAGVETTTPSKQSNNKYAA W SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC SEQ ID NO. 9: HC DESIGN 5.0EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFILSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VK KYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 10:LC DESIGN 5.0 DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQAN D ATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 11: HC minus V_(H) DESIGN 1.0 + 5.0ASTKGPSVFPLAPSSKSTSGGTAALGCLVK K YFPEPVTVSWNSGALTSG VHT T PAVLQSSGLYSLSSF VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFPLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO. 12: C_(L) DESIGN 1.0 +5.0 GQPKAAPSVTLFPPSSEELQAN D ATLVC F ISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEK TVAPTEC SEQ ID NO. 13:AB2133a(-)pG1 (Design AB2.1.3.3a in (-)pG1 HC)EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVA DVNPNSGGSIYNQ EFKGRFILSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVA T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 14: H.4WTAx1κ(anti-Ax1 LC with H.4 V_(L) and wild-type C_(κ))DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ R KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 15: C_(L) DESIGN 2.1.3.2 GQPKAAPSVTLFPPSSEELQANKATLVC FISDFYPGAVTVAWKADSSPV KAGVETTTPSKQSNNKYAA W SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC SEQ ID NO. 16: H.4WTAx1λ (anti-Ax1 LC with H.4 V_(L) andwild-type C_(λ)) DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ R KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 17:  H.4WT(+)Ax1G1 (Design H.4 in (+)Ax1G1 HC)EVQLVESGGGLVQPGGSLRLSCAASGFSLSGSWIHWVR Y APGKGLEWVGWINPYRGYAYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREYSGWGGSSVGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG SEQ ID NO. 18: HC minus V_(H) DESIGN 2.1.3.2 AND 2.1.3.3AASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG V A T GPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO. 19: HC minus V_(H) DESIGN 2.1.3.3ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG V S T GPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO. 20: C_(L) DESIGN 2.1.3.3 AND 2.1.3.3A GQPKAAPSVTLFPPSSEELQANKATLVC YISDFYPGAVTVAWKADSSPV KAGVETTTPSKQNNKYAA W SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC SEQ ID NO. 21: HC DESIGN AEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVA DVNPNSGGSIYNQ EFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 22:LC DESIGN A R IQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 23: HC DESIGN B EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR KAPGKGLEWVA DVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 24:LC DESIGN B DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 25:  HC DESIGN AB EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR KAPGKGLEWVA DVNPNSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 26:LC DESIGN AB R IQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 27:  HC DESIGN 1.0 + 5.0EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VK KYFPEPVTVSWNSGALTSGVHT T PAVLQSSGLYSLSS F VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSEQ ID NO. 28:  LC DESIGN 1.0 + 5.0DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQAN D ATLVC F ISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 29:  HC DESIGN H.4 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR YAPGKGLEWVA DVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSEQ ID NO. 30:  LC DESIGN H.4, H.5, H.6 LAMBDADIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ R KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 31:  HC DESIGN H.5 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR FAPGKGLEWVA DVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSEQ ID NO. 32:  HC DESIGN H.6 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR WAPGKGLEWVA DVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSEQ ID NO. 33:  LC PERTUZUMAB KAPPA (pκ, KAPPA V_(L)/KAPPA C_(L))DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 34:  LC DESIGN AB KAPPA RIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 35: HC DESIGN 2.1.3.3AEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVA T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSEQ ID NO. 36:  LC DESIGN 2.1.3.3aDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVC Y ISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAA W SYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 37:  HC DESIGNS AB2.1.3.3a and AB2.1.3.2EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR K APGKGLEWVA DVNPNSGGSIYNQ EFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVA T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSEQ ID NO. 38:  LC DESIGN AB2.1.3.3a (AB2133apλ) RIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVC Y ISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAA W SYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 39:  HC DESIGN H.42.1.3.3aEVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR Y APGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVA T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSEQ ID NO. 40:  LC DESIGN H.42.1.3.3aDIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ R KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVC Y ISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAA W SYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 41:  HC DESIGN H.4 KAPPADIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ R KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSENRGECSEQ ID NO. 42:  LC DESIGN AB2.1.3.2 RIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVC F ISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAA W SYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 43:  Matuzumab HC (mG1)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDGRYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPGSEQ ID NO. 44:  Matuzumab LC (mλ, V_(L) and lambda C_(L) [C_(λ))DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVT HEGSTVEKTVAPTECSEQ ID NO. 45:  Matuzumab LC (mκ, V_(L) and kappa C_(L) [C_(κ)])DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQQKPGKAPKLLIYDTSNLASGYPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGECSEQ ID NO. 46:  METMAb HC (METG1)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRKAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGSEQ ID NO. 47:  METMAb LC (METλ, kappa V_(L) and lambda C_(L) [C_(λ)])DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHR SYSCQVTHEGSTVEKTVAPTECSEQ ID NO. 48:  METMAb LC (METκ, kappa V_(L) and kappa C_(L) [C_(κ)])DIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQQKPGKAPKILIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 49:  anti-Ax1 HC (Ax1G1)EVQLVESGGGLVQPGGSLRLSCAASGFSLSGSWIHWVRQAPGKGLEWVGWINPYRGYAYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREYSGWGGSSVGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGSEQ ID NO. 50:  anti-Ax1 LC (Ax1λ, kappa V_(L) and lambda C_(L) [C_(λ)])DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQV THEGSTVEKTVAPTECSEQ ID NO. 51:  anti-Ax1 LC (Ax1κ, kappa V_(L) and kappa C_(L) [C_(κ)])DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 52:  (-)pG1 (Pertuzumab IgG1 HC with CH3_K409D, K392D)EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 53: (+)mG1 (Matuzumab IgG1 HC with C_(H)3_D399K, E356K)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRQAPGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDGRYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG SEQ ID NO. 54: (-)METG1 (METMAb IgG1 HC with CH3_K409D, K392D)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVRQAPGKGLEWVGMIDPSNSDTRFNPNFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 55: (+)Ax1G1 (anti-Ax1 IgG1 HC with C_(H)3_D399K, E356K)EVQLVESGGGLVQPGGSLRLSCAASGFSLSGSWIHWVRQAPGKGLEWVGWINPYRGYAYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREYSGWGGSSVGYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESN GQPENNYKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPG SEQ ID NO. 56: AB2133apκ (Pertuzumab LC with AB in V_(L) and  2.1.3.3a in C_(κ)) RIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC Y LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL W STLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 57: H.4WT(+)mG1 (Design H.4 in (+)mG1 HC)QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDGRYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG SEQ ID NO. 58:  H.4WTmλ(Matuzumab LC with H.4 V_(L) and  wild-type C_(λ))DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQ R KPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTH EGSTVEKTVAPTECSEQ ID NO. 59:  H.4WTmκ (Matuzumab LC with H.4 V_(L) and wild-type C_(κ)) DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQ R KPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGECSEQ ID NO. 60:  AB2133a(-)METG1 (Design AB2.1.3.3a in  (-)METG1 HC)EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR K APGKGLEWVG MIDPSNSDTRFNP EFKDRFTISADTSKNTAYLQMNSLRAEDTAVYYCATYRSYVTPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVA T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 61:  AB2133aMETλ(METMAB LC with AB in V_(L) and  2.1.3.3a C_(λ)) RIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQ D KPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKGQPKAAPSVTLFPPSSEELQANKATLVC Y ISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAA W SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC SEQ ID NO. 62:  AB2133aMETκ(METMAB LC with AB in V_(L) and 2.1.3.3a Cκ) RIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQ D KPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC Y LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL W STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 63:  Wild-Type CκRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGECSEQ ID NO. 64:  2133a Cκ RTVAAPSVFIFPPSDEQLKSGTASVVC YLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSL WSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC SEQ ID NO. 65: HC minus V_(H) DESIGN 2133a + A172RASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG V R T GPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO. 66: HC minus V_(H) DESIGN 2133a + V190MASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG V A T G PAVLQSSGLYSLSSM VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO. 67: HC minus V_(H) DESIGN 2133a + V190IASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG V A T G PAVLQSSGLYSLSSI VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO. 68: HC minus V_(H) DESIGN 2133a + A172R + V190MASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG V R T G PAVLQSSGLYSLSSM VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO. 69: HC minus V_(H) DESIGN 2133a + A172R + V190IASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG V R T G PAVLQSSGLYSLSSI VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO. 70:  Cκ 2133a_V133LRTVAAPSVFIFPPSDEQLKSGTASV L C Y LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL W STLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGECSEQ ID NO. 71:  Cκ_2133a_S174Q RTVAAPSVFIFPPSDEQLKSGTASVVC YLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTY Q L WSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC SEQ ID NO. 72:  Cκ_2133a_S174DRTVAAPSVFIFPPSDEQLKSGTASVVC Y LNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTY DL W STLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC SEQ ID NO. 73: Cκ_2133a_V133L_S174Q RTVAAPSVFIFPPSDEQLKSGTASV L C YLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTY Q L WSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC SEQ ID NO. 74: Cκ_2133a_V133L_S174D RTVAAPSVFIFPPSDEQLKSGTASV L C YLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTY D L WSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC SEQ ID NO. 75: (-)PG1_AB2133a_MR (Pertuzumab) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR KAPGKGLEWVA DVNPNSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVR T G PAVLQSSGLYSLSS M VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 76: (-)PG1_AB2133a_IR (Pertuzumab) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR KAPGKGLEWVA DVNPNSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVR T G PAVLQSSGLYSLSS I VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 77: (-)MetG1_AB2133a_MR (MetMAb) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR KAPGKGLEWVA DVNPNSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVR T G PAVLQSSGLYSLSS M VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 78: (-)MetG1_AB2133a_IR (MetMAb) EVQLVESGGGLVQPGGSLRLSCAASGYTFTSYWLHWVR KAPGKGLEWVA DVNPNSGGSIYNQ E FKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVR T G PAVLQSSGLYSLSS I VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN Y D TTPPVLDSDGSFFLYS DLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 79: Pκ_AB2133a_LD (Pertuzumab) R IQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ DKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV L C Y LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY D L W STLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 80:  Pκ_AB2133a_LQ (Pertuzumab) RIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ D KPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV L C Y LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY Q L W STLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 81:  Metκ_AB2133a_LD (MetMAb) RIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQ D KPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV L C Y LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY D L W STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 82: Metκ_AB2133a_LQ (MetMAb) RIQMTQSPSSLSASVGDRVTITCKSSQSLLYTSSQKNYLAWYQ D KPGKAPKLLIYWASTRESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYAYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASV L C Y LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY Q L W STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO 83:  (+)TG1_H4 (Trastuzumab)EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVR Y APGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG SEQ ID NO 84: (+)TG1_H4 (Trastuzumab) + CS EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVR YAPGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG SEQ ID NO 85: (+)TG1_H4 (Trastuzumab) + DR EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVR YAPGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWG R GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG SEQ ID NO 86: (+)TG1_H4 (Trastuzumab) + CS + DR EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRY APGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWG R GTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG SEQ ID NO 87: Tκ_H4 (Trastuzumab) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ R KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO 88:  Tκ_H4 (Trastuzumab) + CSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ R KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPS K EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO 89:  Tκ_H4 (Trastuzumab) + DRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ R KPG D APKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO 90:  Tκ_H4 (Trastuzumab) + CS + DRDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQ R KPG D APKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPS K EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO 91:  (+)PG1 H4WT (Pertuzumab)EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR Y APGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQID NO 92: (+)PG1 H4WT + CS (Pertuzumab) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR YAPGKGLEWVA DVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO 93: (+)PG1 H4WT + DR (Pertuzumab) EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR YAPGKGLEWVA DVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWG R GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO 94: (+)PG1 H4WT + CS + DR (Pertuzumab)EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVR Y APGKGLEWVADVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCAR NLGPSFYFDYWG RGTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPG SEQ ID NO. 95:  PκH4WT + CS (Pertuzumab) DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ RKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPS K EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 96:  Pκ H4WT + DR (Pertuzumab)DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ R KPG D APKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 97:  Pκ H4WT + CS + DR (Pertuzumab)DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQ R KPG D APKLLIYSASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPS K EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 98:  (+)MG1 H4WT + CS QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVRY APGQGLEWIG EFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDGRYFDYWGQGTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG SEQ ID NO. 99: (+)MG1 H4WT + DR QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR Y APGQGLEWIGEFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCAS RDYDYDGRYFDYWG RGTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG SEQ ID NO. 100: (+)MG1 H4WT + CS + DR QVQLVQSGAEVKKPGASVKVSCKASGYTFTSHWMHWVR YAPGQGLEWIG EFNPSNGRTNYNEKFKSKATMTVDTSTNTAYMELSSLRSEDTAVYYCASRDYDYDGRYFDYWG R GTLVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP D S G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQY QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKA KGQPREPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG SEQ ID NO. 101:  MκH4WT + CS DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQ R KPGKAPKLLIYDTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFG QGTKVEIKRTVAAPSVFIFPPSK EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGECSEQ ID NO. 102:  Mκ H4WT + DR DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQ R KPGD APKLLIYD TSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGECSEQ ID NO. 103:  Mκ H4WT + CS + DR DIQMTQSPSSLSASVGDRVTITCSASSSVTYMYWYQR KPG D APKLLIYD TSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSHIFTFGQGTKVEIKRTVAAPSVFIFPPS K EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGECSEQ ID NO. 104:  (+)BHA10G1 H4WT QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVRY APGQGLEWMG WIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSISSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ Y QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG SEQ ID NO. 105: (+)BHA10G1 H4WT + CS QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR Y APGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARSWEGFPYWGQGTTVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSISSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP DS G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ Y QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG SEQ ID NO. 106: (+)BHA10G1 H4WT + DR QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR Y APGQGLEWMGWIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR SWEGFPYWG RGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSISSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ Y QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG SEQ ID NO. 107: (+)BHA10G1 H4WT + CS + DR QVQLVQSGAEVKKPGSSVKVSCKASGYTFTTYYLHWVR YAPGQGLEWMG WIYPGNVHAQYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAR SWEGFPYWG RGTTVTVSSASTKGPSVFPLAP C SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSISSVVTVPSSSLGTQT YICNVNHKPSNTKVDKKVEP DS G DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ Y QSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPR EPQVYTLPPSR KELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKT TPPVL KSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS LSPG SEQ ID NO. 108: (-)TG1 AB2133a (Trastuzumab) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVR KAPGKGLEWVA RIYPTNGYTRYAD E VKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV A T G PAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NY D TTPPVLDSDGSFFLYSD LTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG SEQ ID NO. 109:  BHA10k H4WTDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQ R KPGKAPKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 110:  BHA10k H4WT + CS DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQR KPGKAPKSLIS SASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKRTVAAPSVFIFPPS K EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 111:  BHA10k H4WT + DR DIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQR KPG D APKSLIS SASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 112:  BHA10k H4WT + CS + DRDIQMTQSPSSLSASVGDRVTITCKASQNVGINVAWYQ R KPG D APKSLISSASYRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQYDTYPFTFGQGTKVEIKRTVAAPSVFIFPPS K EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGECSEQ ID NO. 113:  Tκ AB2133a (Trastuzumab) RIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQP D KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVC Y LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL W STLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC

We claim:
 1. A method for producing a fragment, antigen binding (Fab),the method comprising (all residues are numbered according to the Kabatnumbering system): (1) co-expressing in a host cell: (a) a first nucleicacid encoding a heavy chain variable (VH) domain and a human IgG heavychain constant CH1 (CH1) domain, wherein said VH domain comprises atyrosine at residue 39 and an arginine at residue 105, and said CH1domain comprises an aspartic acid at residue 228, a cysteine at position127 and a glycine at position 230; and (b) a second nucleic acidencoding both a light chain variable (VL) domain and a light chainconstant (CL) domain, wherein said VL domain comprises an arginine atresidue 38 and an aspartic acid at residue 42, said CL domain comprisesa lysine at residue 122, wherein each of said VH and VL domains comprisethree complementarity determining regions (CDRs) which direct binding toan antigen; (2) cultivating said host cell under conditions such thatsaid VH and CH1 domains and said VL and CL domains are produced; and (3)recovering from said host cell a Fab wherein said Fab comprises said VHand CH1 domains and said VL and CL domains.
 2. A method for producing afragment, antigen binding (Fab), the method comprising (all residues arenumbered according to the Kabat numbering system): (1) co-expressing ina host cell: (a) a first nucleic acid encoding a heavy chain variable(VH) domain and a human IgG heavy chain constant CH1 (CH1) domain,wherein said VH domain comprises a tyrosine at residue 39 and anarginine at residue 105, and said CH1 domain comprises an alanine atresidue 172 and a glycine at residue 174; and (b) a second nucleic acidencoding both a light chain variable (VL) domain and a light chainconstant (CL) domain, wherein said VL domain comprises an arginine atresidue 38 and an aspartic acid at residue 42, said CL domain comprisesa tyrosine at residue 135 and a tryptophan at residue 176, wherein eachof said VH and VL domains comprise three complementarity determiningregions (CDRs) which direct binding to an antigen; (2) cultivating saidhost cell under conditions such that said VH and CH1 domains and said VLand CL domains are produced; and (3) recovering from said host cell aFab wherein said Fab comprises said VH and CH1 domains and said VL andCL domains.
 3. A method for producing a fragment, antigen binding (Fab),the method comprising (all residues are numbered according to the Kabatnumbering system): (1) co-expressing in a host cell: (a) a first nucleicacid encoding a heavy chain variable (VH) domain and a human IgG heavychain constant CH1 (CH1) domain, wherein said VH domain comprises alysine at residue 39 and a glutamic acid at residue 62, and said CH1domain comprises an aspartic acid at residue 228, a cysteine at position127 and a glycine at position 230; and (b) a second nucleic acidencoding both a light chain variable (VL) domain and a light chainconstant (CL) domain, wherein said VL domain comprises an arginine atresidue 1 and an aspartic acid at residue 38, said CL domain comprises alysine at residue 122, wherein each of said VH and VL domains comprisethree complementarity determining regions (CDRs) which direct binding toan antigen; (2) cultivating said host cell under conditions such thatsaid VH and CH1 domains and said VL and CL domains are produced; and (3)recovering from said host cell a Fab wherein said Fab comprises said VHand CH1 domains and said VL and CL domains.
 4. The method according toclaim 1, wherein said host cell is a mammalian cell.
 5. The methodaccording to claim 1, wherein said human IgG CH1 domain is IgG1 or IgG4isotype.
 6. The method according to claim 1, wherein said CL domain iskappa isotype.
 7. The method according to claim 2, wherein said hostcell is a mammalian cell.
 8. The method according to claim 2, whereinsaid human IgG CH1 domain is IgG1 or IgG4 isotype.
 9. The methodaccording to claim 2, wherein said CL domain is kappa isotype.
 10. Themethod according to claim 3, wherein said host cell is a mammalian cell.11. The method according to claim 3, wherein said human IgG CH1 domainis IgG1 or IgG4 isotype.
 12. The method according to claim 3, whereinsaid CL domain is kappa isotype.